α-, β-, and γ-Synuclein, a novel family of neuronal proteins, has become the focus of research interest because α-synuclein has been increasingly implicated in the pathogenesis of Parkinson's and Alzheimer's disease. However, the normal functions of the synucleins are still unknown. For this reason, we characterized α-, β-, and γ-synuclein expression in primary hippocampal neuronal cultures and showed that the onset of α- and β-synuclein expression was delayed after synaptic development, suggesting that these synucleins may not be essential for synapse formation. In mature cultured primary neurons, α- and β-synuclein colocalized almost exclusively with synaptophysin in the presynaptic terminal, whereas little γ-synuclein was expressed at all. To assess the function of α-synuclein, we suppressed expression of this protein with antisense oligonucleotide technology. Morphometric ultrastructural analysis of the α-synuclein antisense oligonucleotide-treated cultures revealed a significant reduction in the distal pool of synaptic vesicles. These data suggest that one function of α-synuclein may be to regulate the size of distinct pools of synaptic vesicles in mature neurons.