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- [?] Elisha Cuthbert and Scarlett Johansson were each considered for the role of Gwen Stacy.
- In the comics, the symbiote was a flowing sentient liquid ooze. Sam Raimi was very clear on the film's portrayal of the symbiote: he wanted it to have character, but not to resemble a spider or octopus. As portrayed in the film, it possesses a webbing form (it was composed of many separate CGI threads) that gives it a sense of life and an appearance of gripping onto someone's body.
- In the comics, Harry Osborn dies because the Goblin formula he ingested was fatally unstable, and was slowly poisoning him. Sam Raimi changed Harry's death to parallel his father's death in the first film. However, in Harry's final scenes, his pale look could be a sign of the Goblin formula's effects.
- Kirsten Dunst, a natural blonde, plays redhead Mary Jane, while Bryce Dallas Howard, a natural redhead, plays blonde Gwen Stacy.
- Ground corn was used as sand because it reflected well on camera.
- On May 4th, 2007, while promoting the film on [The Tonight Show with Jay Leno], Thomas Haden Church revealed that he broke three knuckles during the subway scene where he swings to punch Spider-Man and ends up punching a chunk of the wall away. Church said that the effects crew had told him that the brick in the middle was fake while the upper and lower ones were real. Unfortunately, the foam brick had not actually been put in place yet, and when Sam Raimi yelled 'action', Church spun around and punched the real brick on the first take.
- Both Topher Grace and Thomas Haden Church confessed that when they were unceremoniously invited to meetings at Sony, they had no idea they would be auditioning for this film.
- According to [?] Grant Curtis, in early production the Vulture was originally going to be in the movie, and Ben Kingsley was involved in negotiations to play him before the character's story line was replaced by Venom.
- In a fight scene where Spider-Man punches through Sandman's chest, congenital amputee boxer [?] Baxter Humby took Tobey Maguire's place in filming the scene. Humby, who was born without his right hand, helped deliver the intended effect of punching through Sandman's chest.
- Eddie's line - "My spider-sense is tingling... if you know what I'm talking about!" - when referring to MJ, was Topher Grace's idea.
- In the comics, Reed Richards of the [Fantastic Four] is the one who discovered that the black and white Spiderman costume is an alien symbiote. However, because the Fantastic Four characters are owned by 20th Century Fox, it was changed to Dr. Curt Connors making the discovery.