The present study aims to investigate the role of extracellular glutamate and NMDA receptor stimulation in the neuronal death induced by a transient exposure to H2O2 of cultured neurons originating from mouse cerebral cortex. Most of the neuronal loss following a transient exposure to H2O2 of cortical neurons results from an apoptotic process involving a secondary stimulation of NMDA receptors, which occurs after H2O2 washout. Indeed, (a) the neurotoxic effect of H2O2 was strongly reduced by antagonists of NMDA receptors, (b) the neurotoxic effect of H2O2 was enhanced in the absence of Mg2+, (c) the protective effect of MK-801 progressively decayed when it was applied with increasing delay time after H2O2 exposure, and (d), finally, the extracellular concentration of glutamate was increased after H2O2 exposure. The major part of H2O2-induced neurotoxicity is mediated by the formation of hydroxyl radicals, which might be involved in (a) the delayed accumulation of extracellular glutamate and NMDA receptor activation and (b) the poly(ADP-ribose) polymerase activation and the related NAD content decrease. The combination of these two mechanisms could lead to both an increase in ATP consumption and a decrease of ATP synthesis. The resulting large decrease in ATP content might be finally responsible for the neuronal death.