According to scholars, the novel's main themes are nature versus nurture, rebirth, and the differing experiences of what society constructs as polar opposites, such as those found between men and women.

Generally, reviewers felt that the novel succeeded in portraying its Greek immigrant drama and were also impressed with Eugenides' depiction of his hometown of Detroit, praising him for his social commentary.

He started writing during his short-term residence at the MacDowell Colony in New Hampshire, United States,[note 2] and finished the novel in Berlin, Germany; he had accepted a grant from the German Academic Exchange Service in 1999.

[14] After discovering in his library research 5-alpha-reductase deficiency, an autosomal recessive condition manifested primarily in inbred, isolated population groups, his perception of the novel significantly changed.

After several years of struggling with the narrative voice, Eugenides finally seated himself at his desk and wrote Middlesex's initial page, "500 words that contained the DNA for the protein synthesis of the entire book.

After learning about the syndrome and facing the prospect of sex reassignment surgery to make her anatomy appear "normally" female, Callie runs away and assumes a male identity as Cal, who hitchhikes cross-country and reaches San Francisco, where he joins a burlesque show as Hermaphroditus.

"[34] [T]he writing itself is also about mixing things up, grafting flights of descriptive fancy with hunks of conversational dialogue, pausing briefly to sketch passing characters or explain a bit of a bygone world.

[44][45] When Cal is baptized as an infant by Father Mike, a Greek Orthodox cleric, the priest receives a surprise: "From between my cherubic legs a stream of crystalline liquid shot into the air ...

[49] According to Penelope Music of Book Magazine, the mismatch in tone of the final two words compared with the rest of the sentence was such that the reading experience was changed from "run-of-the-mill magical realism to true, subversive comedy".

[59] John Mullan, University College London's professor of English and a contributor to The Guardian, wrote that by permitting Cal to be unrealistically aware of fellow characters' thoughts, Eugenides intentionally contravenes an elementary standard in storytelling fiction.

In the novel's closing pages, Cal provides minute details about his father's dying moments and thoughts in a nonsensical car accident even though he is several thousand miles from the scene and only learns of the tragedy from his brother.

[65] The Kirkus Reviews described Middlesex as a "virtuosic combination of elegy, sociohistorical study, and picaresque adventure",[66] and Adam Begley in the New York Observer called it "a hybrid form, epic crossed with history, romance, comedy, tragedy.

[69] Francisco Collado-Rodríguez, a professor of American literature, classified the beginning of Middlesex as a historiographical and metafictional chronicle for its discussion of events such as the Greco-Turkish war and the Great Fire of Smyrna.

[62] Soar posited Desdemona and Lefty's passage as a romantic comedy: the lovers, brother and sister, pretend to be strangers who meet for the first time, attempting "to unknow themselves, to remythologise themselves by developing a past they could live with, unfamiliar and therefore permissible".

The latter half, "full of incest, violence, and terrible family secrets", was considered by Daniel Mendelsohn, an author and critic, to be more effective because Middlesex is largely about how Callie inherited the momentous gene that "ends up defining her indefinable life".

[71] Effectively serving as a double entendre,[72] the title of the book refers to the name of the street where Cal stays at and describes his situation: a hermaphrodite brought up as a girl but who decides to become a boy.

[75] Beginning with Lefty and Desdemona, Cal's grandparents, fleeing from their homeland to Ellis Island and the United States, the novel later depicts the family living in a suburban vista at Grosse Pointe, Michigan.

[25][67][85] Critic Raoul Eshelman noted that despite these conflicts, the narrator is able to achieve "ethnic reconciliation" when he moves to Berlin and lives with the Turks, who had murdered his forebears in the early 20th century and who had indirectly enabled his grandparents to consummate their incestuous relationship.

Soar noted that "for the three generations of Greek Americans who people Middlesex, the mulberry trees of Mount Olympus are an appropriately antique beginning: they are the egg inside which everything began".

In a manner similar to Oedipus's fulfillment of Pythia's prophecy to slay his father and marry his mother, Callie validates the prediction her grandmother made before her birth by adopting a male identity.

[103] When Callie reaches puberty, her testosterone levels increase significantly, resulting in the formation of a larger Adam's apple, the broadening of her muscles, the deepening of her voice, and the augmentation of her clitoris to resemble a penis.

[108] Through Cal, scholar Angela Pattatucci Aragon stated, Eugenides opines that the 1876 system devised by Edwin Klebs that used gonad tissue to determine sex provides the most accurate answer.

Like the masks of Greek drama, Middlesex is equal parts comedy and tragedy, but its real triumph is its emotional abundance, delivered with consummate authority and grace.

"[146] Daniel Mendelsohn of The New York Times Book Review wrote that thematically, there was no reason that a Greek should be an intersex or vice versa and that Eugenides had two disconnected stories to tell.

[44] Clay Risen of Flak Magazine believed that the immigrant experience was the "heart of the novel", lamenting that it minimized the story of Callie/Cal who is such a "fascinating character that the reader feels short-changed by his failure to take her/him further.

[149] Lisa Zeidner of The Washington Post opined that Middlesex "provides not only incest à la Ada and a Lolita-style road trip, but enough dense detail to keep fans of close reading manically busy.

"[151] Paul Quinn of Contemporary Literary Criticism commended the novel, writing: "That Eugenides manages to move us without sinking into sentiment shows how successfully he has avoided the tentacles of irony which grip so many writers of his generation.

"[152] Christina McCarroll of The Christian Science Monitor wrote that "Eugenides wrangles with a destiny that mutates and recombines like restless chromosomes, in a novel of extraordinary flexibility, scope, and emotional depth.

"[99] David Kipen of the San Francisco Chronicle agreed, opining "[a]mong so many other things, this praiseworthy, prize-worthy yarn succeeds as a heartbroken mash note to the Detroit of Eugenides' birth, a city whose neighborhoods he sometimes appears to love—as he loves his characters—less for their virtues than for their defects.

In The Virgin Suicides, Eugenides resplendently portrayed the intense fear during virginal sex, as well as Gabriel García Márquez, the 1982 Nobel Prize in Literature laureate; in Middlesex, the single event in which the novel "comes to life" is Eugenides' depiction of Callie's liaison with her adolescent lover; and in The Marriage Plot, the novel was exceptional in its "sweet banter of courtship" and the "doormat nice-boy role" the character Mitchell assumes in his interplay with his darling, Madeleine.