Many of the Pinky and the Brain episodes occur at Acme Labs, located in a large American city underneath a suspension bridge.

Several episodes take place in historical times, with Pinky and Brain in the laboratory of some scientifically minded person, including Merlin,[3] H. G. Wells,[4] Ivan Pavlov,[5] and Johannes Gutenberg.

[11] Pinky (voiced by Rob Paulsen) is a genetically modified mouse who shares the same cage as Brain at Acme Labs.

And in "The Pink Candidate", when Pinky became president, he later began citing various constitutional amendments and legal problems that would bar Brain from his latest plot to take over the world.

He is highly intelligent and develops complex plans for global domination using politics, cultural references, and his own inventions toward his goal.

[12] Due to his stature and megalomania, Brain has been compared to Don Quixote[13] and has been called a pop culture depiction of Napoleon Bonaparte.

Other characters that have appeared on the series have included both of Brain's[20] and Pinky's parents[21] and the duo's "son", "Roman Numeral I" (Romy for short) another white mouse who was the result of a cloning mistake.

Episodes also include recurring caricatures of celebrities, including both Bill and Hillary Clinton, David Letterman, Dick Clark, Drew Carey, Ryan Stiles, Kathy Kinney, J. D. Wilkes, Paul Gilmartin, Annabelle Gurwitch, Don Rickles, Michael Keaton, Eric and Donny from Too Something, "Macho Man" Randy Savage, The Allman Brothers Band, David Cross, Jack Black, Chris Shiflett, Jim Nabors as Gomer Pyle, and Christopher Walken, as was common on other Animaniacs cartoons.

Animation and developed interesting personalities that the other staff had picked up on; Ruegger said that Minton seemed to be always scheming to take over the world, while Fitzgerald comedically agreed with him, injecting nonsense words like "Narf" and "Egad" around the office.

"[25] During the development of Animaniacs, animator Bruce Timm drew caricatures of Minton and Fitzgerald, and Ruegger then added mouse ears and noses to the drawing, cementing the concept for Pinky and the Brain.

[29] The writers developed the script in secret playing off these test lines and the Frozen Peas outtakes, and arranged to have recording on the same day as Sam Kinison's funeral, whom LaMarche was a close friend of.

Peter Hastings, Rusty Mills and Liz Holzman produced the series when it was spun off from Animaniacs, as well as the season it ran primetime on the WB.

Upon moving into its own series, the writing staff included Gordon Bressack, Charles M. Howell IV, Earl Kress, Wendell Morris, and Tom Sheppard.

The series also used the work of many of the same voice actors for Animaniacs, including Tress MacNeille, Jess Harnell, Frank Welker, Nancy Cartwright, Janet Waldo and Jeff Bennett.

Celebrities such as Roddy McDowall, Nora Dunn, Townsend Coleman, Ernest Borgnine, Eric Idle, Dick Clark, Ed McMahon, Steve Allen, Joyce Brothers, Gavin MacLeod, John Tesh, Michael McKean, Garry Marshall, Mark Hamill, James Belushi, and Steven Spielberg have all performed guest voice work for the series as well.

In the first version, Yakko, Wakko, and Dot (voiced respectively by Paulsen, Harnell, and MacNeille) popped up in the lab and sang the theme while letting the two mice out of their cage.

On the Pinky and the Brain series, the theme gained an additional two verses and was sung by Harnell, Dorian Harewood, Jim Cummings, and Paulsen.

Like Animaniacs, most of the original Pinky and the Brain segments used a variety of animation studios , including Tokyo Movie Shinsha, StarToons, Wang Film Productions, and AKOM.

The bulk of the episodes created outside of Animaniacs (seasons 2 and beyond) were produced by Rough Draft Studios, Wang Film Productions and AKOM.

Parodies of pop culture icons were quite common on the series, more so during the original episodes developed for the WB prime time slot.

"[37] The three-part episode "Brainwashed" included several allusions to the television show The Prisoner, though everyone in this version of the Village was identified by the hat that they wore, and not by a number.

The song includes such odd lyrics as "Put your fingers in your ears, then stick them in your belly" and "Bop yourself on the head and cross your eyes."

For example, "Around the World in 80 Narfs", where the mice are foiled by trying to speak "New York cabbie" and end up going in circles in one location, the gag credit word was "anophelosis" defined as "morbid state due to extreme frustration".

In an interview on the third DVD volume, LaMarche and Paulsen noted that Roy Langbord (vice-president of Showtime), Al Franken, and Barenaked Ladies are fans of the shows.

The episode "Inherit the Wheeze",[47] in which Brain was subject to the effects of smoking by a tobacco company, won a PRISM Award for its anti-smoking message.

The first season was scheduled in a prime time slot from September 10, 1995, through July 21, 1996, as part of the new WB network lineup, with episodes also being repeated within the Saturday morning cartoon block.

[19][27] At this point, Peter Hastings, a key writer for the series, decided to quit, with his last script being "You'll Never Eat Food Pellets in This Town Again!

[27] With increased pressure from the WB network, the series eventually was retooled on September 19, 1998, into Pinky, Elmyra & the Brain, in which Pinky and the Brain were owned by Tiny Toon Adventures character Elmyra Duff; the unusual change in format was even sarcastically noted in the altered title song, with lyrics such as "It's what the network wants, why bother to complain?".

[27] This series lasted for 13 episodes, six of which were shown unedited and seven of which were split up into segments and aired as a part of The Cat&Birdy Warneroonie PinkyBrainy Big Cartoonie Show.

An expanded version of the Animaniacs segment "Bubba Bo Bob Brain" presented in a radio drama or audiobook fashion was released as a read-along book and CD in 1997 by Rhino Entertainment.