In the eponymous anime television series, Misato is head of the operations department of the special agency Nerv, and is in charge of directing and devising war strategies needed to defeat mysterious beings named Angels.

Some critics have contrasted her histrionic personality and sexual overtones with her 14-year-old subordinate and Evangelion protagonist Shinji Ikari; others have appreciated her spontaneity, tenacity, and endearment.

[7] Sadamoto tried to give Misato a "girl next door" role; she was originally thought of as a fashion-conscious woman who constantly changes her clothing and apparently unsuited to military life; she was also conceived as emotional, stubborn and frivolous, and able, if forced by events, to sleep with all of the men at Nerv.

[14] In the twentieth episode, Anno introduced an implied sequence with a long off-screen shot of Misato uttering verses of pleasure and arguing while having sex with her lover, Ryōji Kaji.

[29] According to Mitsuishi, "During scenes where her feelings exploded or she poured out her heart, I also became a bit over-emotional ... My hands shook and it took all my might to keep the script I was holding from rustling and making noise".

[31] For the character's last scene in The End of Evangelion, in which Misato says goodbye to Shinji by leading him into an elevator, Mitsuishi received approval as early as the first cut.

For the third chapter in the saga Evangelion: 3.0 You Can (Not) Redo (2012), which is set fourteen years after the first, Hideaki Anno asked her to act naturally and to be as cold as possible.

In the concluding chapter of the saga Evangelion: 3.0+1.0 Thrice Upon a Time (2021), Anno used theatrical techniques to ensure even in Misato's case the camera did not point at her face in particularly heavy scenes so Mituishi's interpretation would not be affected by her lip-reading.

[45][46] In 2009, after finishing her university studies, Misato joins the German division of a research center called Gehirn and meets Asuka Langley Soryu, a pilot assigned to command the Eva-02.

Misato, shaken by the discovery, begins to doubt the real intentions of the Nerv[63][64] and those of her friend Ritsuko Akagi, director of the technical department of the organization.

[65] In the movie The End of Evangelion, the day she discovers the truth a secret organization called Seele orders the requisitioning of the Nerv headquarters, which is stormed by troops of the Japanese Strategic Self-Defense Forces.

[72] In the second installment of the saga, Evangelion: 2.0 You Can (Not) Advance (2009), Misato appears less cautious and less reserved than in the original version;[73] during the feature film, she pushes Shinji to get closer to his father Gendo and begins to emotionally talk with Asuka.

[75][76] In Evangelion: 3.0 You Can (Not) Redo (2012), set fourteen years after the previous one, Misato plays the role of head of an organization named Wille, which was created to destroy the Nerv, as well as captain of a flying ship called AAA Wunder.

[78] In the final feature film of the saga, Evangelion: 3.0+1.0 Thrice Upon a Time (2021), Misato travels with the crew of AAA Wunder to Antarctica, the epicenter of the Second Impact, to attempt to neutralize a unit called Eva-13.

[29] Hideaki Anno wanted to portray Misato as an eternal teenager who, despite childhood traumas and a difficult experience, begins a process of inner analysis through which she builds her own psychologically acceptable reality, in which the fears of the past coexist with everyday life.

[116][117][118] When Shinji arrives at Nerv's headquarters, Misato immediately sees his potential as a pilot,[119][120] a selfish consideration that later causes her a strong sense of guilt.

[125] After the initial embarrassment, however, Shinji understands Misato's true motives, as she shows him her helpless, rough side because she trusts him and considers him a member of her family.

Misato's relationship with Shinji has been compared to that between a mother and her son; an older sister and a younger brother;[136] an adult and a child; a superior and a subordinate; and between two lovers.

[149] Misato's attitude has been attributed to an Electra complex, a psychological concept formulated by Carl Gustav Jung;[149] she unconsciously looks for her father in the figure of Kaji to fill the void caused by the disappearance of her parent.

[155] In the third episode of the series, Misato discusses Arthur Schopenhauer's hedgehog's dilemma with her friend Ritsuko Akagi, highlighting her views on human relationships and communication,[156][157] according to which growing up is a constant attempt to get closer and further away from each other.

[165] According to the book Neon Genesis Evangelion: The Unofficial Guide, Misato's name contains the Japanese word ミサ (misa), which can be translated as "mass", a possible reference to her dark clothing and the cross-shaped pendant she wears around her neck.

[181] After the conclusion of the series, Misato ranked eighth among the most-popular female characters of the moment in the 1996 Anime Grand Prix, a large annual survey conducted by the Japanese magazine Animage.

[190] Moreover, according to Animerica magazine, Misato's popularity led her to become the most-popular female character in the series in the United States, surpassing Rei Ayanami and Asuka.

[192] Japanese website Niconico ranked her first among animated characters that are most suited to the role of big sister, appreciating both her messy, affectionate, homely attitude and her military skills.

Anime News Network reviewer Kenneth Lee found her characterization ineffective, writing about the lack of convincing explanations about Misato's emergence from her years of aphasia.

[203][204] Comic Book Resources' Ajay Aravind noted the kiss between Shinji and Misato as one of the scenes that changed Japanese animation;[205] his colleague Devin Meenan described it as "one of the most beautiful" of The End of Evangelion.

[209] The website Japanator expresses a contrary opinion, saying it is "fascinating to see Misato unchained from the shackles of Nerv", emphasizing the consistency of the changes and finding them relevant to the spirit of Rebuild of Evangelion.

[211] Comic Book Resources' Reuben Baron also welcomed the revelations presented in the feature film, saying; "3.0+1.01 also fills in enough details about Misato's experiences during the time skip [in 3.0] to give her a more convincing emotional arc".

[231] Critics have found similarities to Misato in later female anime characters, including Talho Yūki from Eureka Seven by her way of drinking beer[232] and Haruka Shitow from RahXephon for her maternal attitude toward the series' protagonist, Kamina.

[235] Her colleague Jason Green similarly traced Misato's influence on the characterization of Yoko Littner from Gurren Lagann, particularly in the use of "Gainax bounce" fan service.