Synaptic vesicles from the electric organ of the marine ray Narcine brasiliensis, purified to at least 90% homogeneity, were analyzed for the lipid and fatty acid content of their membranes. The major lipids (mol %) were phosphatidylcholine (32.3%), phosphatidylethanolamine (20.5%), phosphatidylserine (6.1%), sphingomyelin (3.0%), and cholesterol (33.3%), a composition which did not differ greatly from that of the parent electric organ. While the number of double bonds per fatty acid molecule was similar for both synaptic vesicle and whole electric organ phospholipids, the vesicles were highly enriched in docosahexenoic acid (22:6). Reaction with the amine labeling reagents isethionylacetimidate and trinitrobenzenesulfonic acid indicated that 40% of the phosphatidylserine and 60% of the phosphatidylethanolamine are present on the external (cytoplasmic) surface of the synaptic vesicle. These data on a natural fusing membrane have relevance to models of membrane fusion, which have been based largely on studies of in vitro fusion using synthetic membranes.