Amphitryon belonged to the Perseid royal house of the Argolid. His father was Alcaeus, a son of Perseus and Andromeda. Apollodorus gives several traditions for his mother: Astydameia, daughter of Pelops; Laonome, daughter of Guneus; or Hipponome, daughter of Menoeceus. His sisters included Anaxo and Perimede. Anaxo married Electryon and became Alcmene's mother, while Perimede married Licymnius. In this genealogy Alcmene's mother Anaxo was also Amphitryon's sister. His proposed marriage to Alcmene belonged to the closely linked branches of the Perseid family.

The disaster began with a cattle raid. The sons of Pterelaus claimed a share of the Mycenaean kingdom, and fighting with Electryon's sons left the royal household devastated. Licymnius survived. Amphitryon recovered the stolen cattle, while Electryon prepared to avenge his sons and entrusted the kingdom and Alcmene to him. Before he could depart, a cow strayed and Amphitryon threw a club to turn it back. The club rebounded and struck Electryon, killing him. Sthenelus used the killing to expel Amphitryon and take control. Amphitryon fled with Alcmene to Thebes, where Creon purified him, but she required vengeance for her brothers before they could share a bed.

Creon agreed to support the expedition on one condition: Amphitryon must first rid Thebes of the Teumessian Fox. The animal was destined never to be caught. Amphitryon sought help from Cephalus, whose hound, known as Laelaps, was destined always to catch its quarry. He promised Cephalus a share of the spoils from the coming campaign. The pursuit placed two incompatible destinies against one another: an unfailing hunter and prey that could never be taken. Zeus resolved the paradox by turning both animals to stone. With the threat removed, Amphitryon could gather the allies needed for the voyage against Pterelaus.

The island king possessed a golden hair which protected his life. His daughter Comaetho fell in love with Amphitryon and removed the hair, bringing about her father's death and the defeat of his kingdom. The betrayal helped the invaders succeed, but it did not win Comaetho the marriage she desired. Amphitryon killed her, divided the conquered territory among his allies and returned to Thebes. Comaetho's hope had helped decide the war, but the victory brought her death rather than a place in the conqueror's household. Alcmene's brothers had been avenged, and the condition she had placed on their union had been met.

Before Amphitryon arrived home, Zeus visited Alcmene in his shape and described the completed campaign as though he had fought it. When the real husband returned, she believed that he had already been there. Tiresias explained the deception. Alcmene bore twins: Heracles was the son of Zeus, while Iphicles was Amphitryon's son. Amphitryon nevertheless took his place in the household that raised them both. Apollodorus includes him among Heracles' teachers, assigning him instruction in driving a chariot. The hero's divine parentage did not remove the role of the mortal father who helped bring him up.

In Apollodorus's account, Amphitryon's life ended in the war between Thebes and the Minyans of Orchomenus. Thebes had been forced to pay tribute after the killing of Clymenus, father of the Minyan king Erginus. The young Heracles attacked the tribute collectors, provoking war. The Thebans won and ended their subjection, but Amphitryon died in the fighting. Euripides gives him a different future in Heracles: he survives into old age and witnesses the destruction of his son's household during the hero's madness. These are alternative literary treatments, not stages of one biography. Between them, Amphitryon remains the mortal father whose life is inseparable from a divine son's victories and suffering.