Prothoracicotropic hormones (PTTHs) stimulate synthesis and secretion of ecdysteroids by insect prothoracic glands. In Manduca sexta, PTTH exists as two size variants, small and big PTTH. Experiments were performed to assess the possible role of cyclic AMP in small PTTH signal transduction. cAMP analogs, or agents that increase intracellular cAMP, stimulated ecdysteroidogenesis. Small PTTH enhanced glandular cAMP levels; the rise in cAMP preceded an increase in ecdysteroid secretion. Prothoracic glands accumulated less cAMP when treated with small PTTH than when treated with big PTTH. A phosphodiesterase inhibitor (1-methyl-3-isobutylxanthine) (MIX) increased the amount of cAMP in glands treated with small but not big PTTH, suggesting that glandular phosphodiesterase activity may be elevated in the presence of small PTTH. PTTH-stimulated ecdysteroid secretion was suppressed by a cAMP antagonist (Rp-cAMPS). The effects of small and big PTTH on ecdysteroidogenesis were non-additive. The combined results suggest that cAMP is employed as a second messenger by both prothoracicotropins, and that there may be subtle differences in their respective mechanisms of action.