During the larval feeding period, the growth of the wing imaginal disks of Lepidoptera is dependent on continuous feeding. Feeding and nutrition exert their effect via the secretion of bombyxin, the lepidopteran insulin-like hormone. When larvae stop feeding and enter the wandering stage in preparation for metamorphosis, the control of imaginal disk growth becomes feeding and nutrition-independent. Growth of the wing imaginal disks of non-feeding wandering stage Manduca sexta can be stopped by removal of the brain, indicating that a brain-derived factor is required for continued disk growth. Isolated wing disk growth in vitro requires both 20-hydroxyecdysone (20E) and either brain extract or bombyxin to achieve normal growth. In vitro, brain extracts and synthetic bombyxin have little or no effect in stimulating disk growth, but they greatly enhance the effect of 20E, indicating that 20E and bombyxin act synergistically to modulate growth of the imaginal disk. Brain extract and bombyxin induce a suite of insulin-response events in cultured wing disks, which indicate that bombyxin and 20E act through separate and synergistic pathways. The dose-response to 20E reaches a plateau at about 0.1 microg/ml. Tracheal differentiation of the wing disks can be induced to initiate in vitro by a low concentration of 20E, whereas higher concentrations of 20E only stimulate growth.