The neurotoxicity of glutamate and hypoxia was investigated in vitro on hippocampal neurons, which were obtained from 18-day-old rat fetuses and were maintained for 3 days in culture. Chemically defined medium without glutamate was used and the plating density was low enough that the effect of exogenously added glutamate could be directly evaluated. In the normal culture condition 1 mM glutamate was necessary to cause significant neuronal loss in the following 24 h. In marked contrast, when glutamate was added after subcritical hypoxic stress, a dose of glutamate as low as 10 microM could exhibit neurotoxicity. Administration of MK-801, a selective noncompetitive antagonist of the N-methyl-D-aspartate (NMDA) receptor, could in part reverse this increased susceptibility to low-dose glutamate after hypoxia, although MK-801 could not protect hippocampal neurons from high-dose glutamate. Therefore, both the NMDA receptor and other subclasses of the glutamate receptor may be involved in this neurotoxicity of glutamate. Different mechanisms of glutamate neurotoxicity with high and low doses are discussed. Our results showed that hippocampal neurons exposed to subcritical hypoxia become more vulnerable to glutamate than those without hypoxia. This increased susceptibility is of great interest to understanding the mechanism of slowly ongoing neuronal loss caused by ischemia or epilepsy.