Unstimulated (basal) as well as luteinizing hormone (LH)-promoted progesterone production in collagenase-dispersed hen granulosa cells was inhibited in a dose-related manner by two phenothiazines, trifluoperazine (TFP) and chlorpromazine (CP), both of which are known calmodulin antagonists. Using TFP, the more potent antagonist of the two, it was found that LH-stimulated cyclic AMP production was also suppressed. Moreover, TFP attenuated the steroidogenic effects of both 8-bromo-cyclic AMP and isobutylmethylxanthine but had no effect on the conversion of pregnenolone to progesterone. The inhibitory effects of TFP on steroidogenesis were reversible. It is concluded that phenothiazines inhibit steroidogenesis in ovarian granulosa cells by acting at multiple sites both proximal and distal to cyclic AMP generation without influencing the enzyme complex responsible for the conversion of pregnenolone to progesterone. The results are discussed in relation to calmodulin- and non-calmodulin-mediated actions of phenothiazines.