We have investigated the role of mitochondrial calcium buffering in excitotoxic cell death. Glutamate acts at NMDA receptors in cultured rat forebrain neurons to increase the intracellular free calcium concentration. Although concurrent inhibition of mitochondrial calcium uptake substantially enhanced this cytoplasmic calcium increase, it significantly reduced glutamate-stimulated neuronal cell death. Mitochondrial inhibition did not affect nitric oxide production or MAP kinase phosphorylation, which have been proposed to mediate excitotoxicity. These results indicate that very high levels of cytoplasmic calcium are not necessarily toxic to forebrain neurons, and that potential-driven uptake of calcium into mitochondria is required to trigger NMDA-receptor-stimulated neuronal death.