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| Tobey Maguire | Peter Parker/Spider-Man |
| Kirsten Dunst | Mary Jane Watson |
| James Franco | Harry Osborn |
| Alfred Molina | Dr. Otto Octavius/Doc Ock |
| Rosemary Harris | May Parker |
| J.K. Simmons | J. Jonah Jameson |
| Donna Murphy | Rosalie Octavius |
| Daniel Gillies | John Jameson |
| Dylan Baker | Dr. Curt Connors |
| Bill Nunn | Joseph "Robbie" Robertson |
| Vanessa Ferlito | Louise |
| Aasif Mandvi | Mr. Aziz |
| Willem Dafoe | Norman Osborn/Green Goblin |
| Cliff Robertson | Ben Parker |
| Ted Raimi | Hoffman |
| Elizabeth Banks | Miss Brant |
| Bruce Campbell | Snooty Usher |
| Gregg Edelman | Edelman |
| Elya Baskin | Mr. Ditkovitch |
| Michael Edward Thomas | Man at Fire |
Tormented Peter Parker battles a sinister scientist who uses mechanical tentacles for destructive purposes.
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