Abstract The hypothesis that calcium-dependent mechanisms may be involved in regulating ovine placental steroidogenesis was investigated using chorionic cells isolated by enzymatic digestion. Treatment of the cells with the calmodulin antagonist trifluoperazine (TFP) or pimozide caused a dose-related inhibition of progesterone (P 4) production by 80 per cent (P < 0. 001) at 40 μM TFP and 56 per cent (P < 0.001) at 10 μM pimozide. Moreover, the conversion of25 hydroxycholesterol (25 OH Chol.) to P 4 was impaired in the presence of these compounds. These experiments suggest the involvement of a calcium-calmodulin system in the regulation of ovine placental P 4 synthesis. Interestingly, calcium ionophoreA23187 caused a gradual decline in P 4 secretion and completely blocked it at I μM (P < 0.001) and remains absent even in the presence of 25 OH Chol. In contrast, EGTA increased P 4 secretion (P < 0.01). Further, in the presence of 3 mm EGTA the inhibitory effect of 1,μM A23187 was fully reversed. Taken together these results suggest that extracellular calcium could play a role of negative modulation of P 4 secretion in these cells. The possible involvement of protein kinase C (PKC) was tested using tumorpromoting phorbol ester (PMA) or permeant diacylglycerols (OAG or DOG). These compounds were unable to modify basal P 4 secretion but reduced 25 OH Chol stimulated secretion to basal level. The phorbol ester that was unable to activate PKC had no effect on the metabolism of 25 OH Chol. Thus, PMA and diacylglycerol effects are probably mediated by PKC These data support the hypothesis that PKC activation plays a role in the modulation ofcholesterol side-chain cleavage activity in ovine chorionic cells. These results show that calcium-dependent processes are involved in both positive and negative control of P 4 secretion by ovine placenta. Our results also suggest a role for calmodulin and PKC pathways in modulating this secretion.