OK you knew this was coming: No. 1 is "Wrath of Khan," but what else would it possibly be?
Among Trek fans, "TWOK" is the uncontested king of the heap (and among non-Trek fans it's still regarded as a fine piece of cinema) -- I'm going to recap it quickly because everyone knows the drill already:
1) Best villain. Montalban chews the genetically superior scenery. Every Trek villian is billed as "the best one since Khan..." and it's never true
2) Best Kirk. Director Nicholas Meyer forced William Shatner to do his scenes over and over so he'd be too tired for his typical bombastic schtick.
3) Death of Spock. "It was a helluva thing when Spock died..." - George Costanza
And speaking of "Seinfeld" references:
"TWOK" successfully works the "same but different" angle that I've mentioned. It's the same, in that its a sequel to a "TOS" episode, but it's different in that the look and feel are more like a submarine movie than a sci-fi movie. It's also different in that Kirk, the man who's always "cheated death and patted [himself] on the back for his ingenuity" finally has to deal with defeat: he's getting old, he fails to see himself walking into a trap, he disregards orders and people die because of it, he's confronted with the son he never knew and the "life that could have been, but wasn't," and his best friend dies. We'd never seen this from Kirk before, and seeing him more human also made him more heroic.
Fill in the hyperbole -- this is the benchmark for Trek. Here's the director describing his masterpiece: