In the movie, when Pain and Panic kidnapped baby Hercules and made him drink the potion to turn him mortal, Amphitryon and Alcmene heard them, causing Hercules not to drink the whole potion and keep his godly strength, while Pain and Panic fled to find somewhere to hide. Amphitryon and Alcmene find Hercules, and Alcmene picks up the baby, gently telling him not to cry. Amphitryon and Alcmene believe that the baby has been abandoned, or that the Gods have finally answered their prayer to have a child, and they read Hercules' name on his medal, which they realize shows the symbol of the Gods.
But before they can do anything, Pain and Panic, who have turned themselves into snakes, try to attack Amphitryon and Alcmene, but Hercules manages to grab them, tie them together, and throw them far, far away with his divine strength. Though initially shocked by the infant's power, Amphitryon and Alcmene take Hercules to their home and raise him as their own son while his biological parents watch Hercules grow up from Mount Olympus.
Eighteen years later, when Hercules has grown into a young teenager, he and Amphitryon go into town, where Hercules pulls the cart to with his strength after the family donkey, Penelope, somehow twisted her ankle. Once in town, Amphytryon asks Hercules to stay by the cart, which he initially complies with until he tries to catch a flying disc for a group of boys when he accidentally hits his head into a column causing it to fall. Hercules tries to help by catching the column before it falls, but this causes destruction to the whole town. Led by pottery salesman Demetrius, who finally vents his fury at Hercules and Amphitryon over the destruction of his merchandise and the marketplace, the whole town angrily blames Hercules for the destruction, and Demetrius angrily tells Amphitryon to keep him away from there, adding in the final insult with calling Hercules a "freak", which the rest of the angry town agrees on, leaving Hercules crushed. After this, Amphitryon tells Hercules not to listen to the peoples' comments, but Hercules feels like he really is a freak and begins to question where he really belongs.
Later on, Amphitryon and Alcmene decide to tell Hercules the truth about finding him and show him the medal with his name on it, which has the symbols of the Gods on it. Though initially surprised about the news, Hercules decides to go to the Temple of Zeus and find out where he belongs but tells Amphitryon and Alcmene before he leaves that they're the greatest parents anyone (or he) could have.
The next day Hercules hugs Amphitryon and Alcmene good-bye as he begins his quest to find out where he belongs. During the song "Zero to Hero" Amphitryon and Alcmene are seen buying Hercules souvenirs after Hercules becomes famous for saving the world from monsters, and now have a bigger house. At the end, when Hercules decides to stay on Earth with Meg instead of becoming a God again, he is reunited with Amphitryon and Alcmene and they happily watch as Hercules' biological father Zeus paints a picture of him in the sky for being a true Hero after rescuing Meg from Hades.
Amphitryon and Alcmene remain mostly absent in the series, which takes place after Hercules has left them to train with Philoctetes, but make an appearance in two episodes.
In "Hercules and the Parent's Weekend", Amphitryon and Alcmene make their first appearance in the animated series and have a major role when they both come to visit Hercules during an open house event at Prometheus Academy.
In "Hercules and the Underworld Takeover", Hercules is given the responsibility of looking after his adoptive father's farm while he is recovering from his leg injury.
In "Hercules and the Green-Eyed Monster", Hercules mentions his adoptive parents in attempts to comfort and reassure Icarus who he is upset that his father is planning to remarry.
- In traditional myth, Alcmene was not Hercules' adoptive mother, but his biological mother. Amphitryon was still unrelated to Hercules, though he was his stepfather. Also, in contrast to their portrayal as peasant farmers in the Disney film, the mythological Alcmene and Amphitryon were of royal blood.