The insect prothoracic gland produces ecdysteroids that elicit molting and metamorphosis, and neurohormone stimulation of steroidogenesis by this gland involves both Ca2+ and cyclic adenosine monophosphate second messengers. Prothoracic gland adenylate cyclase exhibits a complex Ca2+/calmodulin (CaM) dependence, a component of which requires an activated Gs alpha for expression. A developmental switch in this system has been identified that correlates with a change in both regulation and function of the gland and involves the loss of sensitivity to extracellular Ca2+ at a time approximately concurrent with the loss of Ca2+/CaM sensitivity by the adenylate cyclase. The extent of cholera toxin activation of gland Gs alpha is lowered before this developmental switch. However, no alterations in Gs alpha levels or mobility are detected, suggesting that Gs alpha interaction with another component in the signaling pathway, perhaps adenylate cyclase itself, produces the apparent Ca2+/CaM dependence and influences the ability of toxin to modify Gs alpha.