Glutamate-induced neurotoxicity has been proposed to depend on a sustained increase of intracellular free Ca2+ levels. However, the molecular mechanism(s) involved are not well understood. Some results suggest that activation of protein kinase C by the increased levels of Ca2+ could play a role in the mediation of glutamate neurotoxicity. To assess this hypothesis we have tested if the 1-(5-isoquinolinyl-sulfonyl)-2-methylpiperazine (H7) and calphostin C, inhibitors of protein kinase C, are able to protect neurons in primary culture from glutamate-induced cell death. It is shown that both H7 and calphostin C prevent nearly completely the death of neurons from cerebellum, even when 2 mM glutamate was used. HA-1004, an inhibitor of cyclic nucleotide-dependent protein kinases, did not protect neurons. The protective effect was maximum at approximately 10 microM H7 and at approximately 10 nM calphostin C. The results reported support the hypothesis that protein kinase C plays a key role in the mediation of glutamate neurotoxicity.