Both prostaglandin F2 alpha (PGF2 alpha) and LHRH inhibit LH-stimulated cAMP accumulation and progesterone secretion in the intact luteal cell, but have no effect on LH-sensitive adenylate cyclase activity in isolated membranes. The present studies were conducted to assess the possibility that calcium (Ca2+) may mediate the inhibitory activity of PGF2 alpha and LHRH in the rat luteal cell. Removal of extracellular Ca2+ significantly enhanced cAMP accumulation in response to LH by about 2-fold, but blunted LH-stimulated progesterone secretion. Incubation of luteal cells with A23187 caused a highly significant and dose-related decrease in LH-stimulated cAMP accumulation with a concentration for half-maximal inhibition (IC50) of about 1 microM. No effect of A23187 was seen on LH-sensitive adenylate cyclase activity, but the ionophore elicited significant inhibition of LH-stimulated intracellular cAMP accumulation in the presence of isobutyl-methylxanthine (MIX), a phosphodiesterase inhibitor. Inhibition by A23187 was Ca2+ dependent, since a decrease in extracellular Ca2+ to less than 100 microM completely blocked the effect of the ionophore. A23187 also significantly inhibited LH-stimulated progesterone secretion in response to LH or cholera toxin and inhibited cholera toxin-stimulated cAMP accumulation in the absence or presence of MIX. In incubations of isolated luteal membranes, Ca2+ produced a dose-dependent inhibition of LH-stimulated adenylate cyclase activity in the absence or presence of MIX at free Ca2+ levels between 5-20 microM (IC50, approximately 10 microM). Depletion of extracellular Ca2+ had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha in the intact cell, and the inhibitory activity of LHRH was slightly reduced, but not abolished, by depletion of extracellular Ca2+. Verapamil, a Ca2+ channel blocker, had no effect on inhibition of LH-stimulated cAMP accumulation by PGF2 alpha or LHRH. It is concluded that an acute increase in intracellular Ca2+ inhibits activation of adenylate cyclase by LH in the rat luteal cell. This conclusion is based on studies that showed enhanced cAMP accumulation by LH in Ca2+-depleted media, Ca2+-dependent inhibition of LH-stimulated cAMP production by a Ca2+ ionophore, and direct inhibition of LH-sensitive adenylate cyclase activity by Ca2+ in luteal membranes. It is suggested that a similar effect occurs in response to PGF2 alpha or LHRH in the luteal cell, but inhibition by these luteolytic agents is not dependent on an influx of extracellular Ca2+, but, rather, is due to an increase in intracellular Ca2+ by other mechanisms.