Protein I is one of the best candidates for a neuronal protein whose phosphorylation may have a functional role in synaptic activity. It is a substrate for both cyclic AMP-dependent and protein kinases, and these kinases show differential specificity for its multiple phosphorylation sites. Protein I is found exclusively in the central and peripheral nervous systems, and immunohistochemical and subcellular fractionation studies suggest an association primarily with synaptic vesicles. Using slices of rat cerebral cortex incubated in vitro, Protein I was phosphorylated both by agents which increase intracellular cyclic AMP and by agents causing Ca2+ influx, although not by any putative neurotransmitters or neuromodulators. We have now examined the facial motor nucleus and report here that serotonin produces a phosphorylation of Protein I when incubated with facial nucleus slices. Demonstration of a neurotransmitter-dependent alteration in the state of phosphorylation of a synapse-specific protein may be due to the relatively simple neuronal circuitry within the facial motor nucleus.