OBJECTIVE The purpose of these studies was to determine the source(s) of the increase in intracellular free calcium in response to luteinizing hormone-releasing hormone in ovarian granulosa cells. METHODS Rat granulosa cells were cultured and loaded with fura-2-acetoxy-methyl ester, a fluorescent calcium indicator dye, and intracellular free calcium measured by microspectrofluorometry. The source of the luteinizing hormone-releasing hormone induced increase in intracellular Ca++ was investigated with various calcium channel blockers (verapamil, diltiazem, and nifedipine), high K+ buffer, and perifusion with media lacking Ca++. RESULTS All three voltage-sensitive calcium channel blockers (10(-5) mol/L) tested were ineffective in blocking the luteinizing hormone-releasing hormone induced intracellular Ca++ increase. Treatment with high K+ buffer also had no effect. Perifusion with media lacking calcium resulted in a gradual loss of the luteinizing hormone-releasing hormone response, an effect that was accelerated by repeated stimulation with hormone. Transient replacement of extracellular Ca++ failed to restore the response but continued perifusion with Ca(++)-replete media allowed a luteinizing hormone-releasing hormone response 10 minutes later. CONCLUSIONS The luteinizing hormone-releasing hormone induced intracellular Ca++ increase does not appear to result from the opening of voltage-sensitive or K(+)-dependent Ca++ channels. To the contrary, this response likely results from the release of Ca++, primarily from intracellular stores.