Emerging evidence indicates that d-serine rather than glycine serves as an endogenous agonist at glycine site of the N-methyl-d-aspartate (NMDA) subtype of glutamate receptors, in several nervous tissues, including the developing cerebellum and the retina. Here, we examined whether endogenous d-serine plays a significant role in neuronal damage resulting from excitotoxic insults in the cerebral cortex, using rat brain slices maintained in a defined salt solution. Neuronal cell death induced by application of NMDA or by oxygen-glucose deprivation (simulated ischemia) was markedly suppressed by a competitive glycine site antagonist 2,7-dichlorokynurenic acid. Addition of glycine or d-serine did not augment neuronal damage by NMDA or simulated ischemia, indicating that sufficient amount of glycine site agonist(s) is supplied endogenously within the slices. Application of d-amino acid oxidase, an enzyme that degrades d-serine, markedly inhibited neuronal damage by NMDA and simulated ischemia, which was reversed by addition of excess d-serine or glycine. Sensitivity to the glycine site antagonist of NMDA- or ischemia-induced damage was not affected by the presence of a non-NMDA receptor antagonist, suggesting that kainate receptor-stimulated d-serine release as demonstrated in primary cultured astrocytes does not contribute significantly to the extent of neuronal injury in these settings. The present results suggest that endogenous supply of d-serine as a glycine site agonist is important for neuronal injury involving NMDA receptor overactivation in the cerebral cortex.