The Brain (voiced by Maurice LaMarche) looks and sounds like Orson Welles, albeit with pink eyes. In "What Ever Happened to Baby Brain", Brain in fact crosses paths with Welles, who is working as a busboy in a Hollywood restaurant; they find themselves inadvertently yelling in unison, "Things will be different when I take over the world!" In "Project B.R.A.I.N." Brain's name is the acronym for "Biological Recombinant Algorithmic Intelligence Nexus." His tail is bent like a staircase—he often uses it to pick the lock of his cage—and his head is large and wide, housing his abnormally large brain. He is highly intelligent and develops complex plans for global domination (only nobody believed he will), using politics, cultural references, and his own inventions toward his goal. He seems coldly unemotional, speaking in a deadpan. Nevertheless, Brain has a subtle sense of humor and even possesses some Christmas spirit. He has even fallen in love, with Trudie in the episode "The Third Mouse", and with Billie in "The World Can Wait." Brain has been compared to Napoleon Bonaparte and Don Quixote.
Brain sees his inevitable rise to power as good for the world, and not mere megalomania. Though it should be noted that despite his malevolent sounding plan he's not really in the typical sense a villain. In Wakko's Wish, he said to Pinky "We're on our way to fame, fortune and a world that's a better place for all." Many of the Brain's plots had the endgame of winning over the people's hearts and having them make him their ruler. However, his motives are not pure. In one episode, Brain finds himself hypnotized by a psychologist he had planned to manipulate for one of his schemes, none other than Sigmund Freud. There Brain reveals that he originally lived with his parents in a tin can. When he was young, researchers took him from his home, and the last he saw of it was a picture of the world on the side of the can. Dr. Freud speculates that Brain's hunger to take over the world stems from wanting to go back home to his parents.
In a crossover episode with Buttons and Mindy, Brain appears as Mindy's pet mouse and tries to take over the world with her help, but things go astray very quickly. In the episode, it appears that Brain can use telekinesis.
Pinky (voiced by Rob Paulsen) is another genetically modified mouse who shares the same cage at Acme Labs, but is much more dim. He speaks with an exaggerated cockney accent. He has several verbal tics, such as "narf", "zort", "poit", and "troz"(the last of which he started saying after noticing it was "zort in the mirror"). Pinky has a straight tail, blue eyes, and a severe overbite, and is taller than the Brain. Pinky's name was given to him by Brain, thinking that Brain was calling him when in fact Brain was referring to his own pinky digit, when insulting some scientists while talking to himself.
Pinky is more open-minded than the Brain and much happier. Troubles never ruin his day, mostly because he is too scatter-brained to notice them. He helps Brain toward world domination, even though Brain insults him and often hits him on the head. Pinky actually seems to enjoy this, laughing after he's hit. He is happy just being with his best friend. He is obsessed with trivia, spending a lot of time watching television and following popular culture fads. This detritus is in the place of the Brain's rigorous logic. However, the Brain's schemes have sometimes tapped into this knowledge. Sometimes Pinky even finds non-rational solutions to problems. He has a number of unusual special abilities, something like 'magic', perhaps caused by his genetic engineering. He occasionally levitates, and also is apparently capable of telekinesis, as seen when he levitated several pieces of fruit in a bowl. 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 process and how he comes to the strange, seemingly non-sensical responses to the Brain's famous question.