The involvement of the calcium-calmodulin system in the gonadotropic regulation of granulosa cell steroidogenesis during follicular development was assessed by comparing the effects of agents known to alter calcium metabolism or calmodulin activity on gonadotropin-stimulated progesterone production by rat granulosa cells at different stages of cellular differentiation. Granulosa cells from pregnant mare's serum gonadotropin-injected rats (PMSG-cells) could be stimulated to produce steroids by both follicle-stimulating hormone (FSH) and luteinizing hormone (LH), whereas those from 17 beta-estradiol-pretreated animals (E2-cells) only responded to to FSH. Although PMSG-cells demonstrated greater capacities for adenosine 3': 5'-cyclic monophosphate (cyclic AMP) and progesterone production, their sensitivity to the various agents tested was similar to that of the E2-cells. [Ethylene-bis(oxyethylene-nitrilo)] tetraacetic acid (EGTA) markedly reduced both basal and FSH-stimulated progesterone production by both cell preparations. Verapamil, an agent which inhibits cellular uptake of calcium, attenuated cyclic AMP and progesterone production by both cell groups at similar concentrations. Calcium ionophore A23187, a stimulator of cellular calcium uptake, significantly stimulated progesterone production by both E2-cells and PMSG-cells. While FSH-stimulated progesterone production by both cell groups was lowered by the calmodulin inhibitor, 1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-beta-(2,4-dichlorobenzy loxy) phenethyl] imidazolium chloride (R24571), cyclic AMP production by PMSG-cells in response to both FSH and LH was significantly reduced by R24571 and trifluoperazine (TFP), another calmodulin inhibitor. These findings indicate that the calcium-calmodulin system is involved in the regulation of granulosa cell steroidogenesis by both FSH and LH. Its participation in the gonadotropic regulation is independent of the stage of follicular maturation and cellular differentiation.