The morphogenetic activity of three hormonal substances—α-ecdysone, β-ecdysone, and inokosterone—has been studied in vitro on isolated imaginal leg discs of third-instar larvae of Drosophila melanogaster. In the presence of α-ecdysone (0.3–3 μg/ml) and also of the phytohormone inokosterone (0.3–3 μg/ml), the discs underwent metamorphosis, as characterized by complete evagination (in less than 24 hr), secretion, and shedding (48 hr after explanation) of the pupal cuticle, secretion, and structural differentiation of the imaginal cuticle, namely pigmentation and formation of claws, bristles, and hairs (during days 3–6). In the presence of β-ecdysone (10, 6, 3, 0.3, 0.03, 0.003 μg/ml), evagination was always abnormal and incomplete. With all concentrations but the lowest, the partially everted legs had a swollen appearance and, at all concentrations, the subsequent development was inhibited. No imaginal differentiation occurred at any of the concentrations tested. Larval fat body or larval epidermis added to the isolated discs had no influence on their response to either α-ecdysone or β-ecdysone. Changing the osmotic pressure of the β-ecdysone containing medium likewise did not alter the noxious effect of β-ecdysone. Discs cultured first in the presence of β-ecdysone (for 24 hr), then transferred to fresh medium containing α-ecdysone were unable to undergo normal development. The inhibitory effect of β-ecdysone thus appears to be irreversible. Discs cultured first in the presence of α-ecdysone (for 24, 48 or 72 hr), then transferred to β-ecdysone containing medium, were unable to continue their normal differentiation. Further development was blocked within a few hours after the transfer. Results are discussed in view of results obtained with other in vitro and in vivo cultivation techniques. In conclusion, isolated leg discs of Drosophila are unable to respond physiologically to exogenous β-ecdysone. Only α-ecdysone and inokosterone will induce complete and normal metamorphosis in leg discs cultured in vitro.