The effects of human chorionic gonadotropin (HCG), forskolin, cyclic nucleotides, the phosphodiesterase inhibitors IBMX and theophylline, cyanoketone, and cycloheximide on the production of estradiol-17β by isolated ovarian follicles of vitellogenic goldfish (Carassius auratus) were examined using 18-hr incubations. HCG and all test agents which are known to increase intracellular concentrations of cAMP significantly stimulated the production of estradiol-17β. However, dibutyryl cGMP was unable to stimulate estradiol-17β production at any concentration used (1–10 mM). Cyanoketone at a concentration of 1 μg/ml completely blocked forskolin-induced estradiol-17β production. Even in the presence of cyanoketone, however, forskolin stimulated conversion of exogenous testosterone to estradiol-17β in a dose-dependent manner, suggesting the involvement of an adenylate-cyclase system in the induction of aromatase activation by vitellogenic follicles of goldfish. Cycloheximide also completely abolished HCG-induced estradiol-17β production when this inhibitor was added within the first 1 hr after the addition of HCG. These results provide evidence that the stimulation of estradiol-17β by goldfish vitellogenic follicles in response to HCG is dependent upon the synthesis of new protein.