Depolarization-induced calcium influx into rat cerebral cortex synaptosomes increased the phosphorylation of several synaptosomal proteins as examined by 32Pi incorporation. A phosphopeptide mapping technique involving NaDodSO4/polyacrylamide gels has been used to show that phosphorylation of a Mr 87,000 substrate protein is stimulated by depolarization-induced calcium influx. Phosphorylation of this Mr 87,000 substrate occurred in synaptosomal cytosol and was markedly stimulated by calcium/phosphatidylserine. Calmodulin inhibited this phosphorylation reaction. This substrate for calcium/phospholipid-dependent protein kinase is enriched in and appears to be specific to neurons.