To elucidate the sequence of molecular events leading to nerve terminal differentiation, we have examined the regulation of expression of presynaptic protein genes during synapse formation in vivo. In the chick ciliary ganglion (CG), synaptophysin IIa and synaptophysin IIb mRNAs showed threefold increases relative to neurofilament-M mRNA during the time of target contact [Embryonic Day 7 (E7)-E9]. Expression of synaptotagmin I mRNA also increased severalfold over this time interval. Thus, mRNAs for three synaptic vesicle proteins are upregulated coordinately during synaptogenesis. In contrast, the major increase in choline acetyltransferase (ChAT) mRNA (four- to fivefold) occurred between E15 and E20, coincident with the maturation of synapses in the CG. Coincident with ChAT upregulation, there is a switch in the relative abundance of mRNAs encoding vesicle protein isoforms. In particular, mRNAs encoding synaptophysin IIb and synaptotagmin II (which is undetectable at E9) become predominant. Therefore, although synaptic vesicle protein mRNAs are upregulated in a first phase of differentiation at the time of synapse formation, a temporally distinct phase of presynaptic protein gene regulation, associated with the specific maturation of synapses, is also apparent.