Ecdysteroid production by the prothoracic glands of the tobacco hornworm, Manduca sexta was found to be under feedback control by the ecdysteroids in hemolymph using both culture in vivo in diapausing pupae lacking the brain-corpora cardiaca-corpora allata complex and the prothoracic glands and culture in vitro. Prothoracic glands having relatively high activity in larvae, prepupae, or developing pupae were inhibited by ecdysone or 20-hydroxyecdysone. By contrast, prothoracic glands with low activity from feeding larvae, day 1 non-diapausing pupae and diapausing pupae were activated by both ecdysone and 20-hydroxyecdysone in vivo and in vitro. Dose-response studies on diapausing pupal glands showed that ecdysone was the most effective activator. These findings suggest that prothoracic glands are either stimulated or inhibited by ecdysone or 20-hydroxyecdysone, depending on both the secretory activity of the gland and the effective level of ecdysteroids in hemolymph. Thus, when the glands are first activated, the ecdysteroids that are secreted show a positive feedback on the glands to increase ecdysteroid output. Then the activated glands are turned off by the increasing 20-hydroxyecdysone titer in the hemolymph leading to the rapid decrease in ecdysteroid titer at the end of the molt period.