alpha -Latrotoxin (LTX) stimulates vesicular exocytosis by at least two mechanisms that include (1) receptor binding-stimulation and (2) membrane pore formation. Here, we use the toxin mutant LTX super(N4C) to selectively study the receptor-mediated actions of LTX. LTX super(N4C) binds to both LTX receptors (latrophilin and neurexin) and greatly enhances the frequency of spontaneous and miniature EPSCs recorded from CA3 pyramidal neurons in hippocampal slice cultures. The effect of LTX super(N4C) is reversible and is not attenuated by La super(3+) that is known to block LTX pores. On the other hand, LTX super(N4C) action, which requires extracellular Ca super(2+), is inhibited by thapsigargin, a drug depleting intracellular Ca super(2+) stores, by 2-aminoethoxydiphenyl borate, a blocker of inositol(1,4,5)-trisphosphate-induced Ca super(2+) release, and by U73122, a phospholipase C inhibitor. Furthermore, measurements using a fluorescent Ca super(2+) indicator directly demonstrate that LTX super(N4C) increases presynaptic, but not dendritic, free Ca super(2+) concentration; this Ca super(2+) rise is blocked by thapsigargin, suggesting, together with electrophysiological data, that the receptor-mediated action of LTX super(N4C) involves mobilization of Ca super(2+) from intracellular stores. Finally, in contrast to wild-type LTX, which inhibits evoked synaptic transmission probably attributable to pore formation, LTX super(N4C) actually potentiates synaptic currents elicited by electrical stimulation of afferent fibers. We suggest that the mutant LTX super(N4C), lacking the ionophore-like activity of wild-type LTX, activates a presynaptic receptor and stimulates Ca super(2+) release from intracellular stores, leading to the enhancement of synaptic vesicle exocytosis.