Expression screening was used to isolate cDNA clones encoding a synaptic vesicle membrane protein, VAT-1, which is specifically expressed in the electric lobe of marine rays. The predicted protein has a molecular weight of 41,572 daltons and contains several hydrophobic regions. An antibody raised against a fusion protein synthesized in E. coli recognizes an abundant 42 kd protein that copurifies largely with synaptic vesicles. Trypsin digestion of intact and lysed vesicles as well as membrane extractions suggests that VAT-1 is an integral membrane protein. The VAT-1 RNA is localized to the electromotor nucleus, and the fusion protein antibody stains the electric organ, demonstrating that the protein is transported to nerve terminals. These studies define a novel synaptic vesicle protein that is likely to play a central role in the functions mediated by specific classes of synaptic vesicles.