Alpha-synuclein is a neuronal protein implicated both in synaptic transmission and in neurodegenerative diseases. Although it is evident that this protein is enriched in the presynaptic terminals of neurons, localization in synaptic vesicles has not been conclusively determined. Here, we show that alpha-synuclein is present, but not enriched, in synaptic vesicles using highly purified synaptic vesicle preparations from rat brain homogenate. Immunoisolation of vesicles using antibodies against synaptophysin or synaptobrevin confirmed the presence of alpha-synuclein in synaptic vesicles. Additional separation of synaptic vesicles by sucrose velocity centrifugation showed that there are different subpopulations of synaptic vesicles and that alpha-synuclein is present only in a specific subpopulation, whereas synaptophysin and synaptobrevin were found in all the synaptic vesicles. Presence of alpha-synuclein only in a subset of synaptic vesicles suggests that this protein may have a specific function in synaptic vesicle cycling, hence in synaptic transmission.