Larval Manduca prothoracic gland cells in vitro responded to prothoracicotropic hormone (PTTH) from neurosecretory cells of the brain with an increase of intracellular free calcium. This effect is reversible and dose-dependent. Preincubation of the glands with TMB-8 and dantrolene, which inhibit the release of calcium from intracellular stores, did not decrease the PTTH-stimulated increase in calcium, indicating that intracellular calcium stores are not involved in the control of ecdysteroidogenesis. Pharmacological studies of the PTTH effect with calcium channel blockers revealed that the increase in calcium was totally blocked by cadmium, partially inhibited by nickel and lanthanum and by amiloride, an antagonist of T-type calcium channels. All other inhibitors tested were ineffective, suggesting that the increase in cytosolic calcium is induced by opening of calcium channels, presumably of the T-type, in response to PTTH. The action of PTTH on these channels may be mediated by a G-protein as shown by the effect of mastoparan, a G-protein activator, which increased the concentration of cytosolic calcium comparable to that evoked by PTTH., Heiner Birkenbeil, and Lit