Antibodies directed against the lumenal domain of synaptotagmin I conjugated to CY3 (CY3-Syt1-Abs) and video microscopy were used to study the dynamics of synaptic vesicles in cultured hippocampal neurons. When applied to cultures after synapse formation, CY3-Syt1-Abs produced a strong labeling of presynaptic vesicle clusters which was markedly increased by membrane depolarization. The increase of the rate of CY3-Syt1-Ab uptake in a high K+ medium was maximal during the first few minutes but persisted for as long as 60 min. In axons developing in isolation, CY3-Syt1-Abs, in combination with electron microscopy immunocytochemistry, revealed the presence of synaptic vesicle clusters which move in bulk in anterograde and retrograde direction. Clusters are present both in the axon shaft and in filopodia but not in the filopodia of the growth cone. Both presynaptic vesicle clusters and clusters present in isolated axons were disrupted by okadaic acid as previously shown for synaptic vesicle clusters at the frog neuromuscular junction. These findings indicate that synaptic vesicle aggregation may occur independently of cell-cell interaction, but that, in the absence of a synaptic contact, vesicle clusters are not stably anchored to a given region of the cell surface. Labeling of synaptic vesicles in immature isolated neurons was found to be depolarization and Ca2+ dependent, demonstrating that Ca(2+)-regulated exocytosis is an intrinsic characteristic of synaptic vesicles irrespective of their localization at a synapse.