Alpha-latrotoxin (LTX) from the black widow spider venom, stimulates neurotransmitter release from neuronal cells via Ca2+ -dependent as well as Ca2+ -independent mechanisms. In some peptide-secreting endocrine cells, however, LTX stimulates hormone release mainly via a Ca2+ -independent mechanism. Here we investigated the action of LTX in rat pituitary gonadotropes that secrete the peptide, LH. Using the patch-clamp technique in conjunction with the fluorescent Ca2+ indicator (indo-1) to simultaneously measure the cytosolic Ca2+ concentration ([Ca2+]i) and ionic current, we showed that LTX elicited bursts of inward current that were accompanied by [Ca2+]i elevations. In the presence of a physiological concentration of extracellular Ca2+, the unitary conductance of the LTX-induced current was about 300 pS, and only about 6.4% of the current was carried by Ca2+. The LTX-induced current was occasionally followed by intracellular Ca2+ release. At [Ca2+]i of 1 microM or more, exocytosis (detected by membrane capacitance measurement) was consistently triggered, and it was frequently followed by endocytosis. Thus, LTX triggers Ca2+ -dependent exocytosis in gonadotropes via extracellular Ca2+ entry as well as intracellular Ca2+ release. In approximately 25% of the cells, LTX could also trigger a slow exocytosis in the absence of [Ca2+]i elevation. Therefore, LTX has both Ca2+ -dependent and Ca2+ -independent actions in gonadotropes.