We studied the effect of alpha-latrotoxin (alpha LTX) on [14C]acetylcholine ([14C]ACh) release, intracellular Ca2+ concentration ([Ca2+]i). plasma membrane potential, and high-affinity choline uptake of synaptosomes isolated from guinea pig cortex. alpha LTX (10(-10)-10(-8) M) caused an elevation of the [Ca2+]i as detected by Fura 2 fluorescence and evoked [14C]ACh efflux. Two components in the action of the toxin were distinguished: one that required the presence of Na+ in the external medium and another that did not Displacement of Na+ by sucrose or N-methylglucamine in the medium considerably decreased the elevation of [Ca2+]i and [14C]ACh release by alpha LTX. The Na(+)-dependent component of the alpha LTX action was obvious in the inhibition of the high-affinity choline uptake of synaptosomes. Some of the toxin action on both [Ca2+]i and [14C]ACh release remained in the absence of Na+. Both the Na(+)-dependent and the Na(+)-independent components of the alpha LTX-evoked [14C]ACh release partly required the presence of either Mg2+ or Ca2+. The nonneurotransmitter [14C]choline was released along with [14C]ACh, but this release did not depend on the presence of either Na+ or Ca2+, indicating nonspecific leakage through the plasma membrane. We conclude that there are two factors in the release of ACh from synaptosomes caused by the toxin: (1) cation-dependent ACh release, which is related to (a) Na(+)-dependent divalent cation entry and (b) Na(+)-independent divalent cation entry, and (2) non-specific Na(+)- and divalent cation-independent leakage.