Granulosa cells from estrogen-treated immature rats were incubated in chemically defined media containing FSH, cholera toxin, (Bu)2AMP, and/or 3-isobutyl-1-methyl-xanthine in the presence and absence of a calcium chelator (EGTA), an inhibitor of uptake of extracellular calcium [verapamil or lanthanum (La)], or an inhibitor of calmodulin [trifluoperazine or 1-[bis-(p-chlorophenyl)methyl]3-[2,4-dichloro-beta-(2, 4-dichlorobenzyloxy)phenethyl]imidazolium chloride]. Regardless of the presence of 3-isobutyl-1-methyl-xanthine, FSH stimulated cAMP and progesterone production. La inhibited the basal and FSH-stimulated synthesis of progesterone and gonadotropin-enhanced cAMP production. Whereas the net synthesis of cAMP was also inhibited by La in the presence of 3-isobutyl-1-methyl-xanthine, it was increased in the absence of the phosphodiesterase inhibitor. EGTA decreased the basal, FSH-stimulated, and cholera toxin-stimulated production of progesterone but not of cAMP. While (Bu)2cAMP stimulated progesterone production, this response was markedly attenuated by La, verapamil and EGTA. Addition of the calmodulin inhibitors to the granulosa cell incubations also markedly decreased the FSH-stimulated production of cAMP and progesterone as well as the steroidogenic response to the dibutyryl cyclic nucleotide. These findings suggest that calcium plays an important role in the regulation of progesterone production in the rat granulosa cell. In addition to its requirement in the control of cellular cAMP levels, calcium may be involved at a step(s) in the steroidogenic pathway distal to the cAMP cascade.