The effects of arachidonic acid and phorbol esters in the Ca(2+)-dependent release of glutamate evoked by 4-aminopyridine (4-AP) in rat cerebrocortical synaptosomes were studied. In the absence of arachidonic acid, high concentrations (500 nM) of 4 beta-phorbol dibutyrate (4 beta-PDBu) were required to enhance the release of glutamate. However, in the presence of arachidonic acid, low concentrations of 4 beta-PDBu (1-50 nM) were effective in potentiating glutamate exocytosis. This potentiation of glutamate release by phorbol esters was not observed with the methyl ester of arachidonic acid, which does not activate protein kinase C. Moreover, pretreatment of synaptosomes with the protein kinase inhibitor staurosporine also prevented the stimulatory effect by arachidonic acid and phorbol esters. These results suggest that the activation of protein kinase C by both arachidonic acid and phorbol esters may play a role in the potentiation of glutamate exocytosis.