The involvement of cAMP in the steroidogenic action of the cerebral neuropeptide, prothoracicotropic hormone (PTTH), was examined in the tobacco hornworm, Manduca sexta. PTTH stimulates ecdysone synthesis by paired organs in the thorax, the prothoracic glands. The steroidogenic effect of PTTH on prothoracic glands of day 3 fifth instar larvae and day 0 pupae is mimicked in vitro by agents that act by increasing the intracellular levels of cAMP (1-methyl-3-isobutylxanthine, cAMP analogs and forskolin). In addition, partially purified big PTTH (Mr 28500 daltons) stimulates the formation of cAMP in both stages of glands in a rapid, dose-dependent manner. However, a significant accumulation of cAMP in response to PTTH occurs only in larval prothoracic glands. In pupal glands, effects of the neuropeptide on cAMP synthesis are seen only in the presence of a phosphodiesterase inhibitor. The results indicate that cAMP is involved as a second messenger in PTTH action, but that the developmental status of the prothoracic glands influences the degree to which cAMP accumulates in response to the neurohormone.