Prothoracicotropic hormone (PTTH) is the primary regulator of ecdysone biosynthesis by insect prothoracic glands. In Manduca sexta, a stimulatory protein from larval hemolymph appears to be an important secondary regulator of gland activity. A third level of regulation may involve the competency of the glands to respond to these effectors. To investigate this possibility, glands were removed from larvae on each day of the fifth instar and incubated in vitro, either alone or in the presence of PTTH, the hemolymph protein, or both. Glands were refractory to stimulation by either effector alone, or to a combination of both for the first 2 days of the instar. Subsequent to this, the glands were responsive to either factor alone, but the patterns of responsivity to PTTH and the stimulatory protein were different. After day 2, glands stimulated by a combination of both effectors exhibited a uniform maximal response. The prothoracic glands are, therefore, regulated not only by neuroendocrine and humoral effectors, but also by the competency of the glands to respond to those effectors. © 1986.