Pinky and the Brain is an American animated sitcom created by Tom Ruegger for the Kids' WB programming block of The WB, as a collaboration of Steven Spielberg with his production company Amblin Entertainment and Warner Bros. Television Animation. This was the first animated television series to ever be presented in Dolby Surround. The characters first appeared in 1993 as a recurring segment on the animated television series Animaniacs. It was later spun off as a series due to its popularity, with 65 episodes produced. The characters later appeared in the series Pinky, Elmyra & the Brain, and later returned to their roots as an Animaniacs segment in the 2020 revival of that series.

Pinky and The Brain are genetically enhanced laboratory mice who reside in a cage in the Acme Labs research facility. Pinky is good-natured but feeble-minded, while The Brain is highly intelligent but arrogant and bitter. In each episode, The Brain devises a new plan to take over the world which ultimately ends in failure, usually due to the impossibility of The Brain's plan, The Brain's own overconfidence, Pinky's bumbling, an oversight on The Brain's part, circumstances beyond their control, or a combination thereof. In common with many other Animaniacs shorts, many episodes are in some way a parody of something else, usually a film or novel.

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, H. G. Wells, Ivan Pavlov, and Johannes Gutenberg. There is very little continuity between episodes, outside of the common fixtures of the two mice. Though some plans for world domination from early episodes are subsequently referred to in later seasons; for example, Brain's mechanical human suit that was first used in "Win Big" reappears when Brain faces his rival Snowball in "Snowball".

The bulk of every episode involves one of Brain's plans for world domination with Pinky's assistance and the ultimate failure of that plan, with some exceptions. One centers on Snowball's plan to take over the world using Microsponge (a parody of Microsoft). Another episode features Brain's single day where he tries to do anything but take over the world: in the end, a group of people votes that he should take over the world on the one day that he does not want to.

Both Pinky and Brain, white lab mice kept as part of Acme Labs' experimentation, have undergone significant genetic alteration, per the show's title lyrics, "their genes have been spliced" which gives the two mice amplified intelligence over that of a typical mouse, the ability to talk to humans, and anthropomorphism. "Project B.R.A.I.N." suggests that the gene splicing occurred on September 9, 1995, which is coincident to the first full episode of Pinky and the Brain. The episode "Brainwashed" states that the gene splicing was done by Dr. Mordough, along with Snowball the hamster and Precious the cat, using the Acme Gene Splicer, a bagel warmer, and a hot dog steamer.

Although Pinky and Brain plan to conquer the world, they do not show much animosity. In a Christmas special, Pinky even wrote a letter to Santa Claus saying that Brain had the world's best interests at heart.

Pinky (voiced by Rob Paulsen) is a genetically modified mouse who shares the same cage as Brain at Acme Labs. Pinky is an extremely unstable and hyperactive mouse. He has several verbal tics, such as "Narf", "Zort", "Poit", and "Troz", the last of which he started saying after noticing it was "Zort" reflected backwards in a mirror. Pinky's appearance contrasts with Brain's – while Brain is short, has a crooked tail and pink sclerae, and speaks in a deeper, more eloquent manner, Pinky has a straight tail, blue sclerae, and a severe overbite. Pinky is taller than Brain and speaks in a high-pitched voice with a Cockney British accent. Pinky's name was inadvertently given to him by Brain himself: when insulting the two scientists responsible for their gene splicing while talking to himself, Brain claimed the scientists had "less knowledge in both their heads than I do in my...pinky!" Pinky then responded with "Yes?", believing that Brain was referring to him.

Pinky is more open-minded, kinder, and generally happier than Brain. Troubles never ruin his day, possibly because he is too scatter-brained to notice them. He steadfastly helps Brain toward world domination, even though Brain usually berates, belittles and even abuses him. Pinky actually seems to enjoy this, often laughing after he is hit on the head. He is obsessed with trivia, spending a lot of time watching television in the lab and following popular culture fads. Sometimes, Pinky even finds non-rational solutions to problems. An entire episode (entitled "The Pinky P.O.V.") even shows a typical night of attempted world domination from his point of view, showing his thought processes and how he comes to the strange, seemingly nonsensical responses to Brain's famous question, "Pinky, are you pondering what I'm pondering?" Pinky often points out flaws in the Brain's plans, which the Brain consistently ignores. The issues Pinky brings up can ironically lead to the downfall of the plan in any given night's plot.