Upon treatment with sodium carbonate, rat brain synaptic vesicles lost ATP-dependent H+ transport and released major polypeptide components (about 72, 57, 41, 34 and 33 kDa). These polypeptides, consisting about 15% of the total protein, were identified as subunits of H(+)-ATPase by immunoblotting with antibodies against H(+)-ATPase from chromaffin granules. The same treatment also abolished the ATP-dependent, bafilomycin-sensitive uptakes of glutamate, serotonin and gamma-aminobutyrate by the synaptic vesicles. These results indicated that H(+)-ATPase is a major constituent of the vesicles (consisting about 20% of their total protein) and is a primary pump for accumulation of neurotransmitters.