The release of acetylcholine (Ach) from Torpedo synaptic vesicles has been investigated. Factors have been found which induce Ca +2 dependent Ach release from the synaptic vesicles. In the absence of these factors, the vesicles are not affected by Ca +2. Addition of a soluble factor to the vesicles induces a Ca +2-dependent release of their Ach. This secretion is enhanced by a non-vesicular membranous component which, by itself, does not induce the Ca +2-dependent release. These results demonstrate that vesicular Ach release may be studied in vitro and thus will enable the study, at the molecular level, of the biochemical events underlying neurotransmission.