Synaptosomes isolated from rat hippocampus contained the alpha- and beta-subspecies of protein kinase C (PKC), but not the gamma-subspecies, whereas postsynaptic pyramidal cells contained all these three subspecies. The PKC enzymes were activated synergistically by diacylglycerol and cis-unsaturated fatty acids including docosahexaenoic acid which is abundant in brain phospholipids. The phosphorylation of a PKC-specific substrate, growth-associated protein (GAP-43), which is associated predominantly with presynaptic membranes, was also protein (GAP-43), which is associated predominantly with presynaptic membranes, was also stimulated by diacylglycerol and cis-unsaturated fatty acids, particularly at the micromolar range of Ca2+ concentrations. The results presented suggest that cis-unsaturated fatty acids together with diacyglycerol may take part in the phosphorylation of GAP-43 in presynaptic membranes presumably by the alpha- or beta-subspecies of PKC even after the Ca2+ concentrations return to the basal level.