As a first step toward elucidating mechanisms involved in the sorting of synaptic vesicle proteins in neurons, we have used immunofluorescence microscopy to determine the distribution of two synaptic vesicle proteins, synapsin I and synaptophysin, in hippocampal neurons developing in culture. In mature cultures, synapsin I and synaptophysin immunoreactivity was concentrated in puncta that were restricted to sites where axons contacted neuronal cell bodies or dendrites. Electron-microscopic immunocytochemistry demonstrated that these puncta corresponded to vesicle-filled axonal varicosities that were exclusively presynaptic. At early stages of development, before cell-cell contact, both synapsin I and synaptophysin were preferentially localized in axons, where they were particularly concentrated in the distal axon and growth cone. In axons that did not contact other cells, immunostaining for these two proteins had a granular appearance, which persisted for at least 7 d, but focal accumulations of vesicles comparable to those seen at sites of synaptic contact were not observed. When neurons contacted one another, numerous puncta of synapsin I and synaptophysin formed within the first week in culture. Double-label immunofluorescence demonstrated that the two vesicle antigens were closely codistributed throughout these stages of development. These observations demonstrate that synaptic vesicle proteins assume a polarized distribution within nerve cells beginning early in development, as soon as the axon can be identified. In contrast, differences in microtubule polarity orientation that distinguish mature axons and dendrites, and that have been proposed to account for the selective sorting of some materials in nerve cells, first appear at a subsequent stage of development. The selective distribution of synaptic vesicle proteins to the axon occurs in isolated cells, independent of interactions with other cells. In contrast, the formation of large clusters of vesicles typical of presynaptic specializations requires contact with an appropriate postsynaptic target. Thus, in cultured hippocampal neurons, the localization of synaptic vesicles in presynaptic specializations is the result of sorting mechanisms intrinsic to individual neurons as well as to mechanisms mediated by cell-cell contact.