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Prothoracicotropic hormone (PTTH) stimulates ecdysteroid secretion by the prothoracic glands of Manduca sexta in a cAMP-dependent manner. However, larval and pupal glands differ markedly in the degree to which PTTH stimulates cAMP accumulation, suggesting a stage-specific difference in phosphodiesterase activity. The present study was designed to determine if and when such a difference arose during development, and its effect on PTTH-stimulated ecdysteroid secretion. The results reveal that soluble phosphodiesterase activity in the prothoracic glands changes significantly during the course of the fifth (last) larval instar, with a marked increase in activity occurring at the onset of prepupal development. Phosphodiesterase activity, particularly in the soluble cell fraction, is inversely correlated with PTTH-stimulated cAMP accumulation. Hormone-stimulated ecdysteroid secretion does not require cAMP accumulation, but does appear to require detectable cAMP synthesis as measured in the presence of phosphodiesterase inhibitors. The amount of ecdysteroid secreted, however, is not proportional to the amount of cAMP synthesized but rather is more closely correlated with developmental changes in glandular protein content.