We have investigated the endocrine mechanisms responsible for the dramatic prolongation of larval life that is associated with imaginal disc overgrowth caused by recessive lethal mutations of the dlg, fat, lgd, c43 and dco genes in Drosophila. dlg, lgd and dco mutant larvae grow at the normal rate during the normal feeding period of the last larval instar, then their growth slows and stops; eventually they reach nearly double the wet weight of pupariating wild-type larvae. fat and c43 mutant larvae do not grow beyond the maximum size seen in wild type. The ecdysteroid titer in all of the mutants remains close to the minimal wild-type level of 3–15 ng/g body wt, and fails to show the sharp peak of about 150 ng/g that is associated with pupariation in the wild type. The main forms of ecdysteroid are free ecdysone and 20-hydroxyecdysone in fully grown larvae of both wild type and the two tested mutants dlg and dco. The secretory activity of dlg and dco ring glands in vitro is about 1/6 that of wild type. The mutant imaginal discs overgrow when cultured in the growth-permissive environment of an adult female abdomen, whereas wild-type discs terminate growth at approximately the normal final size under such conditions. Therefore, the imaginal disc overgrowth is caused by defective function of the mutant gene within the disc itself, and is not a secondary effect of the prolonged larval life. When imaginal disc overgrowth is prevented by γ-irradiation of young dlg larvae, the normal profile of ecdysteroid titers in the larva is restored, and the ecdysteroid production of mutant ring glands explanted and assayed in vitro is elevated. The results support the hypothesis that delayed pupariation in the mutants is caused by the imaginal disc overgrowth and suggest that growing imaginal discs can inhibit the production or release of ecdysteroids from the endocrine system. This might reflect the operation of an important control system that coordinates the termination of imaginal disc growth with the onset of metamorphosis in normal holometabolous insects.