To investigate the involvement of cyclic AMP phosphodiesterase in the antigonadotrophic actions of prostaglandin F2 alpha (PGF2 alpha), human granulosa cells were cultured in serum-supplemented medium for 72 h, treated for 30 min with cloprostenol or phorbol myristate acetate (PMA) and then exposed to human chorionic gonadotrophin (hCG) +/- isobutyl-methylxanthine (IBMX) for a further 4 h. In the absence of IBMX, cloprostenol and PMA inhibited hCG-stimulated progesterone production. However, in the presence of 0.5 mM IBMX, inhibition of phosphodiesterase prevented these antigonadotrophic effects. Phosphodiesterase activity was assessed by a novel direct assay performed on intact cells after 3 days of culture. PGF2 alpha, cloprostenol and 4 beta-PMA all enhanced cAMP degradation whilst an inactive phorbol ester (4 alpha-PMA) had no effect. Down-regulation of protein kinase C by 4 beta-PMA pre-treatment prevented the subsequent stimulation of phosphodiesterase activity by both cloprostenol and 4 beta-PMA. We conclude that the antigonadotrophic actions of PGF2 alpha in cultured human granulosa cells involve a stimulation of cAMP phosphodiesterase mediated via protein kinase C.