Arachidonic acid (AA), a cis-unsaturated fatty acid that activates certain subspecies of protein kinase C (PKC), has been proposed to act as a retrograde messenger in modifying the efficacy of synapses during long-term potentiation (LTP). One prominent PKC substrate of the nerve terminal membrane, GAP-43 (F1, B-50, neuromodulin), shows an increase in phosphorylation that correlates with the persistence of LTP. The present study investigated whether AA might exert its effects on presynaptic endings by modulating the phosphorylation of GAP-43 and other membrane-bound proteins. Using synaptosomal membranes from the rat cerebrocortex, in which in vivo relationships between protein kinases and their native substrates are likely to be preserved, we found that in the absence of Ca2+, AA exerted a modest effect on the phosphorylation of GAP-43 and several other proteins; however, when AA was applied in conjunction with Ca2+, GAP-43 showed a particularly striking response: at Ca2+ levels likely to exist at the nerve terminal membrane during synaptic activity (10(-7) to 10(-5) M), AA (50 microM) increased the sensitivity of GAP-43 phosphorylation to Ca2+ by an order of magnitude, and increased its maximal level of phosphorylation by 50%. At resting Ca2+ levels, AA potentiated the stimulation in GAP-43 phosphorylation produced by 4 beta-phorbol 12,13-dibutyrate, a diacylglycerol (DAG) analog. The stimulatory effect of AA and its synergistic interaction with Ca2+ were found to be mediated by PKC, since they were blocked by a specific peptide inhibitor of PKC, [Ala25]PKC(19–31), but were unaffected by an inhibitor of protein phosphatase activity or by scavengers of free radicals. Since GAP-43 has been implicated in the development and plasticity of synaptic relationships, the synergistic effects of AA and the intracellular signals Ca2+ and DAG on the phosphorylation of GAP-43 may serve as an AND gate to modify presynaptic function and/or structure in response to coincident pre- and postsynaptic activity.