Bitten by a genetically altered spider while visiting Dr. Curt Connors' lab at Empire State University, Peter Parker donned a spider themed costume to fight crime after allowing a burglar to escape, who later killed his Uncle Ben.
Peter was awarded an internship for Dr. Connors, a position which he held until being fired by Martha Connors for ditching the antidote plan to take pictures of the Lizard. However, his Lizard pictures caught the eye of J. Jonah Jameson who gave him a job as a Bugle freelancer to take more pictures of Spider-Man in action. He and Harry try out for the football team, but lets Harry join the football team. In the episode "Catalysts" he goes with Mary Jane to the Fall Formal. However, a few days later, Peter sees Mary Jane going out with Flash Thompson as she says they were just friends.
Following John Jameson's return from space, the alien symbiote was taken to Dr. Connors' lab for study. The symbiote then got loose during a fight between Spider-Man and Black Cat, leading to Spider-Man stepping on and bonding with the symbiote. It causes him to be more aggressive towards his enemies and others; it also gives him a strong desire to keep the symbiote, despite what it is doing to him. After a brief talk with Flash Thompson, Peter is able to think clearly once more and decides to get rid of the symbiote, only to find out it is trying everything it can to bond with him. With the spirit of Uncle Ben to guide him through, Peter was able to triumph over the suit and dispose of it. He later returns it Dr. Connors' lab and tries to destroy the suit forever by freezing it. Unfortunately, Eddie came in at the wrong time and misunderstood the situation, causing his hatred for both Peter and Spider-Man to rise up more than ever, giving the symbiote more than enough energy to break free of the ice and bond with him.
After returning to his normal suit, Peter meets a new villain, Venom. Realizing it is the symbiote and from its words, he realizes it is going after his loved ones. Upon hearing it call him "bro", he finds out that it is Eddie who has bonded with the symbiote. After it nearly attacked Aunt May it swore to get the "person he loved most" and kidnapped Gwen (though Peter assumed he was referring to Mary Jane). Peter decides enough is enough and fights Venom, only to realize he is no match for Venom, Peter tricked the symbiote off of Eddie by pretending to accept it back. Inside of his mind, the symbiote attempted to rejoin with him, only to be stopped by images of Peter's friends and family. Afterwards, Peter buries the symbiote in quick dry cement, deeply hoping never to see it again. Also, by the end of the first season, Peter fully and happily accepts his destiny as Spider-Man, and begins to realize his true feelings for Gwen after she kissed him.
In the second season, Peter realizes his feelings for Gwen and on several occasions tries to talk to her, but his duties as Spider-Man continue to interfere. On New Year's, Liz Allen kisses him revealing that she wants to be with him. The two became a couple later on.
In the episode "Final Curtain", Peter finally talked to Gwen and both confessed their feelings to each other, and agreed to end their respective relationships, which caused him to break up with Liz. Later he discovered the Green Goblin is revealed to be Norman Osborn, he took one of his pumpkin bombs and placed it in his glider, which caused it to explode and crashed with Norman in a water tank filled with pumpkin bombs. Feeling responsible for the death of Norman, Peter felt guilty and decided that he shouldn't be with Gwen and allowed her to stay with Harry.