Intrafollicular luteinization stimulator was shown to be secreted by granulosa cells in culture with stimulatory effects on differentiation of immature granulosa cells. The purpose of this study was to evaluate the role of calcium ions in luteinization stimulator-enhanced luteinization process of granulosa cells. We examined the direct effect of ionophore A23187, voltage-sensitive Ca2+-channel blockers verapamil, nimodipine, nifedipine, niludipine and calmodulin antagonist trifluoroperazine on progesterone and cGMP levels in 3-day culture of small granulosa cells. It was shown that the dihydropyridine derivatives of calcium blocker drugs (nimodipine, nifedipine, niludipine) and calmodulin antagonist (trifluoroperazine) in the micromolar range, significantly suppressed FSH-induced progesterone synthesis and cGMP accumulation in granulosa cells. On the contrary, phenylalkylamine calcium blocker verapamil and calcium ionophore A23187 had different effects on both processes. Calcium ionophore A23187 markedly enhanced cGMP formation, but simultaneously inhibited the FSH-induced progesterone synthesis. Verapamil at lower concentrations (10 jutA) stimulated and at higher concentrations (50 ptA) inhibited the formation of cGMP. To evaluate the role of extra- and intracellular calcium in luteinization stimulator-enhanced progesterone production by small granulosa cells, the effects of indicative agents on stimulatory activity of follicular fluid from large follicles, granulosa cells-conditioned media and granulosa cell extracts were tested. While verapamil is shown to be a less potent modulator, administration of other calcium antagonists as well as ionophore A23187 caused a significant decrease in stimulatory action of follicular fluid from large follicles, granulosa cells-conditioned media and extracts. These findings indicate that the stimulatory action of luteinization stimulator depends on the transport of calcium ions through voltage- sensitive calcium channels and is modulated by alteration of intracellular calcium levels.