According to Billy West, the cast of Star Trek had such good chemistry and were so used to working together that they recorded all their dialogue in the same room and producer David X. Cohen didn't mind.
Two original Star Trek actors are not in this episode. DeForest Kelley, who played Bones, had died 3 years prior. His character appears but doesn't speak. According to the DVD commentary, James Doohan, who played Scotty in the original series, was asked to do the show but said, "No, thank you." For this reason, the episode contains several in-jokes lampooning him, the most obvious being the creation of the fat, unintelligible "Welshie".
When Fry takes Leonard Nimoy from the head museum, the new head that takes his place ("Yes! Front row!") is that of Jonathan Frakes, who, like Nimoy in Star Trek (1966), played the Enterprise's first officer in Star Trek: The Next Generation (1987).
William Shatner's final line - "Let's get the hell out of here." - is a reference to a similar line in Star Trek (1966). The usage in Star Trek is credited as the first time the word "hell" was used on network television. Reportedly, Shatner fought to keep this line in the original show.
At the end, Melllvar quotes a line from Star Trek (1966) to which Fry responds, "episode 10, Balance of Terror." Melllvar then corrects Fry saying, "More like episode 9, loser!" Although Balance of Terror (1966) was the 14th episode aired, it was the ninth episode produced, making Melllvar correct.