alpha-Latrotoxin stimulates neurotransmitter release probably by binding to two receptors, CIRL/latrophilin 1 (CL1) and neurexin I alpha, We have now produced recombinant alpha-latrotoxin (Ltx(WT)) that is as active as native alpha-latrotoxin in triggering synaptic release of glutamate, GABA and norepinephrine, We have also generated three alpha-latrotoxin mutants with substitutions in conserved cysteine residues, and a fourth mutant with a four-residue insertion. All four alpha-latrotoxin mutants were found to be unable to trigger release. interestingly, the insertion mutant Ltx(N4C) exhibited receptor-binding affinities identical to wild-type Ltx(WT), bound to CL1 and neurexin I alpha as well as Ltx(WT), and similarly stimulated synaptic hydrolysis of phosphatidylinositolphosphates. Therefore, receptor binding by alpha-latrotoxin and stimulation of phospholipase C are insufficient to trigger exocytosis, This conclusion was confirmed in experiments with La3+ and Cd2+. La3+ blocked release triggered by Ltx(WT), whereas Cd2+ enhanced it, Both cations, however, had no effect on the stimulation by Ltx(WT) of phosphatidylinositolphosphate hydrolysis, Our data show that receptor binding by alpha-latrotoxin and activation of phospholipase C do not by themselves trigger exocytosis, Thus receptors recruit alpha-latrotoxin to its point of action without activating exocytosis, Exocytosis probably requires an additional receptor-independent activity of alpha-latrotoxin that is selectively inhibited by the Ltx(N4C) mutation and by La3+.