1. Who was the first villain to return for a second battle against Spidey?
The Vulture, who first appeared in Amazing Spiderman #2, returned in Amazing Spiderman #7. Michael Rebain, Kyle, Thelonius Nick and Kryp44 all got this one correct.
2. Name the first villain(s) to appear in three different stories (not issues) in Spiderman.
The first villains to appear in three different stories in Spiderman were The Enforcers: Fancy Dan, Montana, and The Ox. They were henchmen for the Big Man (Fredrick Foswell) in ASM #10, assisted the Green Goblin in ASM #14, and collaborated with the Sandman in ASM #19. For the first time, my commenters were stumped by this question. (Note: You can make an argument for several characters appearing in ASM #18--Goblin, Doc Ock, Vulture, etc., but these were cameos, one-panel appearances). Ironically, ASM 19 was also the last appearance of the three Enforcers with the exception of a cameo flashback in ASM #40.
Clay points out AMS Annual #1 features third appearances of Doc Ock and the Vulture. It is clear that ASM Annual #1 is intended to take place sometime before ASM #18 from a panel in the latter mag. This would also put the Sandman's third appearance in Spiderman ASM #18, or is it in ASM Annual #1, because there was a Sandman appearance in Strange Tales where Spidey helped out.
But I specified in Spiderman, and I meant ASM, not the Annuals. But yeah, this question is pretty slippery.
3. It is well-known that Dr Doom was the first crossover villain in Spiderman. What team was the second?
The Ringmaster and his Circus of Crime originally appeared in the Incredible Hulk #3, and crossed over into ASM #16. Michael Rebain, Kyle, Thelonius Nick and Kryp 44 all got this one correct.
4. Uncle Ben Parker was the first character to die in a Spiderman story. Who was the second?
Bennett Brant, Betty's older brother, bites it in this scene from ASM #11:
Kyle and Kryp44 got this one correct.
5. What guest character appeared the most often in the first 20 issues of Spiderman?
Michael Rebain, Kyle, Thelonius Nick and Kryp 44 all correctly guessed the Human Torch, who appeared in ASM #1, 8, 17, 18 and 19.