The formation of chemical synapses between hippocampal neurons in primary cell culture was studied using electrophysiology, calcium imaging, and immunocytochemical approaches. Inhibitory and excitatory synapses formed within 12 d in cell culture (DIC) that were sensitive to the N-type calcium channel blocker ω-conotoxin GVIA (ω-CgTx). At 4 DIC, immature connections were present in which spontaneous, but rarely evoked, synaptic currents were detected. At both 4 and 12 DIC, the synaptic proteins rab3a, synapsin I, and synaptotagmin were present in hippocampal neurons, but the subcellular distribution changed from one in which immunoreactivity was initially distributed within soma and neurites to a punctate varicose appearance. Correlated with the transformation from immature to mature synaptic states was the onset of ω-CgTx-sensitive calcium influx. Taken together, these data suggest that the expression of functional ω-CgTx-sensitive calcium influx is temporally coincident with synapse formation, and that during the maturation of the synapse there is a redistribution of synaptic proteins.