Excitotoxicity through stimulation of N-methyl-d-aspartate (NMDA) receptors contributes to neuronal death in brain injuries, including stroke. Several lines of evidence suggest a role for protein kinase C (PKC) isoforms in NMDA excitotoxicity. We have used specific peptide inhibitors of classical PKCs (α, β, and γ), novel PKCs δ and ε, and an atypical PKCζ in order to delineate which subspecies are involved in NMDA-induced cell death. Neuronal cell cultures were prepared from 15-day-old mouse embryos and plated onto the astrocytic monolayer. After 2 weeks in vitro the neurons were exposed to 100 µm NMDA for 5 min, and 24 h later the cell viability was examined by measuring the lactate dehydrogenase release and bis-benzimide staining. While inhibitors directed to classical (α, β, and γ) or novel PKCs (δ or ε) had no effect, the PKCζ inhibitor completely prevented the NMDA-induced necrotic neuronal death. Confocal microscopy confirmed that NMDA induced PKCζ translocation, which was blocked by the PKCζ inhibitor. The NMDA-induced changes in intracellular free Ca2+ were not affected by the peptides. In situ hybridization experiments demonstrated that PKCζ mRNA is induced in the cortex after focal brain ischemia. Altogether, the results indicate that PKCζ activation is a downstream signal in NMDA-induced death of cortical neurons.