The involvement of protein kinase C in long-term potentiation was investigated in the mossy fiber-CA3 pathway in an in vitro slice preparation of rat hippocampus. Tetanic stimulation induced stable long-term potentiation in the mossy fiber-CA3 pathway which was not affected by N-methyl-D-aspartate receptor antagonists. Long-term potentiation was not induced in the presence of a protein kinase C inhibitor, sphingosine. Application of 1 microM phorbol-12, 13-diacetate, an activator of protein kinase C, potentiated the synaptic response by about 400% and this potentiation was completely reversible upon washing. Sphingosine blocked the potentiation when it was applied before protein kinase C activation by phorbol-12, 13-diacetate. However, sphingosine had no effect on the potentiation when it was applied after the synaptic response was potentiated to a plateau following phorbol-12,13-diacetate perfusion. Long-term potentiation and phorbol ester-induced potentiation were not additive when phorbol-12,13-diacetate was applied after induction of long-term potentiation, suggesting that long-term potentiation and phorbol-12, 13-diacetate activate the same protein kinase C pool. The enhanced response caused by phorbol-12,13-diacetate returned to the long-term potentiation level after wash-out of phorbol-12,13-diacetate. Thus the cellular changes underlying long-term potentiation are long-lasting or permanent, while those caused by phorbol-12,13-diacetate are not. However, if tetanic stimulation was induced during prolonged phorbol-12,13-diacetate application (1 h), a potentiation similar in amplitude to long-term potentiation was induced but the population response returned to the control pre-long-term potentiation level after 2 h of washing. The potentiation following tetanic stimulation during prolonged application of phorbol-12,13-diacetate was blocked in the presence of D-2-amino-5-phosphonovaleric acid, a N-methyl-D-aspartate receptor antagonist. Thus, in the presence of phorbol esters the N-methyl-D-aspartate-independent long-term potentiation is occluded but a transient potentiation appears, presumably due to hyperexcitability and activation of N-methyl-D-aspartate receptors in recurrent pathways of area CA3. Normal N-methyl-D-aspartate-independent long-term potentiation could be induced after the 2 h washout period and now was maintained. In conclusion, protein kinase C activation is essential but not sufficient for long-term potentiation in the mossy fiber-CA3 pathway and when stimulated by application of phorbol esters produces a large and reversible synaptic potentiation. These investigations show that long-term potentiation in CA3 is a complex event involving several steps, and that activation of protein kinase C is only one of them.