In the opening for the show, Brain is writing a complex formula about the Universe Explained (simplified) and the resulting answer is THX 1138 - the title of the first film by George Lucas.
A bootleg tape of a short-tempered (and foul-mouthed) Orson Welles arguing with a recording engineer during a voice-over session has been widely distributed. It was used as the basis for an episode of this show, with The Brain reading cleaned-up versions of Welles' rantings. The episode's title, "Yes, Always", is taken from one of Orson's complaints.
The Brain's voice is based upon that of Orson Welles. The voice actor portraying Brain is Maurice LaMarche, who also portrayed Orson Welles by dubbing 'Vincent D'Onnofrio' 's role as Orson Welles in the movie Ed Wood (1994).
Pinky and the Brain's genes were spliced using the Acme Bagel Warmer and Gene Splicer.
During the song "Meticulous Analysis of History", The Brain flashes two subliminal messages on the screen. They each appear on the screen for only a fraction of a second. They read, respectively: "You see, you are under my power", and "I forced you to use the 'still' button on your VCR".
Originated as a series of segments on "Animaniacs" before being spun off to its own show. It was moved to the WB's primetime lineup where it won an Emmy. Then it was moved back to the Kids WB lineup where it was "Pinky, Elmyra and the Brain" adding the familiar human character from "Tiny Toon Adventures" (1990). After a few episodes, it was cancelled.