During insect metamorphosis, the steroid hormone 20-hydroxyecdysone (20E) is responsible for coordinating the differentiation of adult structures. Several structures of the Drosophila melanogaster adult leg, the six distalmost joints, the bristles, and the pretarsal claws, were examined to investigate how 20E controls their development in vitro. Joints, bristles, and claws were dependent on 20E for differentiation between 20-22 and 24-26 h after puparium formation (APF). After 26-28 h APF, differentiation became hormone independent. Tissue-specific markers in 20E-free cultures showed that the bristle and joint cells had not undergone any further morphogenetic progression. In contrast, the pretarsi underwent partial differentiation. The concentration of 20E required for differentiation was structure specific; tarsal joints required higher concentrations of 20E (greater than 400 ng 20 E/ml) than pretarsal claws, bristles, and other joints (greater than 40 ng 20E/ml). The 20E precursor ecdysone (E) was also able to induce differentiation at concentrations over 700 ng E/ml, but did not show any synergistic interactions with 20E. Lastly, leg structures had a finite ability to respond to 20E; tarsal joints lost competence to respond after 32-34 h APF, while the remaining structures became incompetent after 44-46 h APF.