Protein I is a neuronal phosphoprotein associated primarily with synaptic vesicles. Regulation of its state of phosphorylation has been investigated in slices of rat facial nucleus. This brainstem motor nucleus has a facilitatory serotonergic input and contains no interneurons. Serotonin (5-hydroxytryptamine, 5-HT, 10(-4) M), in the presence of the phosphodiesterase inhibitor isobutylmethylxanthine (IBMX, 4 x 10(-5) M), converted approximately 26% of Protein I in these slices from the dephospho-form to the phospho-form. This effect was partially inhibited using two classical 5-HT antagonists, mianserin added to the slices during in vitro incubation and metergoline administered in vivo. The effect of 5-HT appeared to be Ca2+-dependent, unlike that of IBMX (10(-3) M). Adenosine, its analog 2- chloroadenosine, and ATP also increased the phosphorylation of Protein I in facial nucleus slices. 2-Chloroadenosine (5 x 10(-4) M) caused a 29% phosphorylation of Protein I, and this effect was not dependent on extracellular Ca2+. The phosphorylation of Protein I caused both by 2- chloroadenosine and by ATP was inhibited by the adenosine antagonist 2′- deoxyadenosine. Results of additional experiments suggest that the great majority of the Protein I in the facial nucleus is present in presynaptic terminals other than the serotonergic afferents. It is concluded that the stimulation by 5-HT and adenosine of Protein I phosphorylation results largely from a direct action of these compounds on those Protein I-containing terminals.