The calcium-dependence of ecdysone synthesis by the insect prothoracic glands was examined in vitro using glands from day 0 pupae of the tobacco hornworm, Manduca sexta. Stimulation of ecdysone synthesis by the cerebral neuropeptide, prothoracicotropic hormone (PTTH), requires extracellular calcium; peptide-stimulated steroidogenesis is blocked by omission of calcium or by addition of the calcium antagonist lanthanum. By contrast, basal synthesis of ecdysone is not calcium-dependent. A stimulatory, as opposed to simply a permissive, role for calcium is indicated by the ability of the calcium ionophore A23187 to mimic the steroidogenic effects of PTTH. Agents that act by increasing the intracellular levels of cAMP (dibutyryl cAMP, 1-methyl-3-isobutylxanthine, forskolin) enhance ecdysone synthesis equally well in the presence or absence of extracellular calcium, indicating that the site of action of the cation in the steroidogenic pathway occurs prior to that of cAMP. Both PTTH and A23187 enhance the formation of cAMP, as measured by the conversion of [3H]ATP to [3H]cAMP, in a manner absolutely dependent upon the presence of extracellular calcium. The results suggest sequential roles for calcium and cAMP in PTTH-stimulated steroidogenesis by the insect prothoracic glands. A model is presented in which the peptide stimulates cAMP formation in a calcium-dependent manner, with the cyclic nucleotide in turn enhancing ecdysone synthesis.