An enhancement of glutamate release from hippocampal neurons has been implicated in long-term potentiation, which is thought to be a cellular correlate of learning and memory. This phenomenom appears to be involved the activation of protein kinase C and lipid second messengers have been implicated in this process. The purpose of this study was to examine how lipid-derived second messengers, which are known to potentiate glutamate release, influence the accumulation of intraterminal free Ca2+, since exocytosis requires Ca2+ and a potentiation of Ca2+ accumulation may provide a molecular mechanism for enhancing glutamate release. The activation of protein kinase C with phorbol esters potentiates the depolarization-evoked release of glutamate from mossy fiber and other hippocampal nerve terminals. Here we show that the activation of protein kinase C also enhances evoked presynaptic Ca2+ accumulation and this effect is attenuated by the protein kinase C inhibitor staurosporine. In addition, the protein kinase C-dependent increase in evoked Ca2+ accumulation was reduced by inhibitors of phospholipase A2 and voltage-sensitive Ca2+ channels, as well as by a lipoxygenase product of arachidonic acid metabolism. That some of the effects of protein kinase C activation were mediated through phospholipase A2 was also indicated by the ability of staurosporine to reduce the Ca2+ accumulation induced by arachidonic acid or the phospholipase A2 activator melittin. Similarly, the synergistic facilitation of evoked Ca2+ accumulation induced by a combination of arachidonic acid and diacylglycerol analogs was attenuated by staurosporine. We suggest, therefore, that the protein kinase C-dependent potentiation of evoked glutamate release is reflected by increases in presynaptic Ca2+ and that the lipid second messengers play a central role in this enhancement of chemical transmission processes.