α-Latrotoxin (α-LTX) is a neurotoxin that accelerates spontaneous exocytosis independently of extracellular Ca2+. Although α-LTX increases spontaneous transmitter release at synapses, the mechanism is unknown. We tested the hypothesis that α-LTX causes transmitter release by mobilizing intracellular Ca2+in frog motor nerve terminals. Transmitter release was measured electrophysiologically and with the vesicle marker FM1-43; presynaptic ion concentration dynamics were measured with fluorescent ion-imaging techniques. We report that α-LTX increases transmitter release after release of a physiologically relevant concentration of intracellular Ca2+. Neither the blockade of Ca2+release nor the depletion of Ca2+from endoplasmic reticulum affected Ca2+signals produced by α-LTX. The Ca2+source is likely to be mitochondria, because the effects on Ca2+mobilization of CCCP (which depletes mitochondrial Ca2+) and of α-LTX are mutually occlusive. The release of mitochondrial Ca2+is partially attributable to an increase in intracellular Na+, suggesting that the mitochondrial Na+/Ca2+exchanger is activated. Effects of α-LTX were not blocked when Ca2+increases were reduced greatly in saline lacking both Na+and Ca2+and by application of intracellular Ca2+chelators. Therefore, although increases in intracellular Ca2+may facilitate the effects of α-LTX on transmitter release, these increases do not appear to be necessary. The results show that investigations of Ca2+-independent α-LTX mechanisms or uses of α-LTX to probe exocytosis mechanisms would be complicated by the release of intracellular Ca2+, which itself can trigger exocytosis.