Synaptosomes were challenged by veratridine, ouabain and alpha-latrotoxin, and the release of 14C-acetylcholine (ACh) was measured in the absence of external Ca2+. We wished to test whether Ca2+ mobilized from internal stores triggered the ACh release that was independent of external Ca2+. We found that none of the agents altered the [Ca2+]i in a Ca(2+)-free medium. Buffering the intracellular Ca2+ concentration with BAPTA did not prevent the increase in release of 14C-ACh by veratridine or ouabain in the absence of Ca2+, however, it greatly reduced the release evoked in a Ca(2+)-containing medium. In parallel samples the release of ACh and the change in the internal Na+ concentration ([Na+]i) were measured. It was found that veratridine, ouabain and alpha-latrotoxin all enhanced [Na+]i in a concentration-dependent manner and a good quantitative relationship existed between the increase in [Na+]i and the release of ACh.