AbstractRoles and mechanisms ofN‐methyl‐D‐aspartate (NMDA) receptors in glutamate neurotoxicity were investigated in cultures of NMDA receptor‐deficient cortical neuronal cells. Mutant mice lacking a functional NMDA receptor were generated by gene targeting of the NR1 NMDA receptor subunit. Cortical neuronal cells prepared from wild‐typeNR1+/+, heterozygousNR1+/‐and homozygous mutant NR1‐/‐mice at 15–17 days of gestation grew indistinguishably from each other. Brief exposures (5 min) of bothNR1+/+andNR1+/‐neuronal cells to glutamate or NMDA, but not kainate or α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionate (AMPA), resulted in widespread neuronal degeneration by the following day. In contrast, neither glutamate nor NMDA treatment caused neuronal degeneration inNR1‐/‐cells, indicating that NMDA receptors are responsible for rapidly triggered glutamate neurotoxicity. The above four compounds were all effectivein inducing the death ofNR1+/+andNR1+/‐neuronal cells after prolonged exposure (20–24 h). However, NMDA had no neurotoxic effects onNR1‐/‐ cells, although the other three compounds were neurotoxic with potencies comparable to those forNR1+/+andNR1+/‐cells. The AMPA and kainate receptors are thus sufficient for inducing slowly triggered glutamate neurotoxicity. Brief exposure of a mixed population ofNR1+/+andNR1‐/‐neuronal cells to NMDA selectively killed the NMDA receptor‐expressing cells without any appreciable effects on neighbouring NMDA receptor‐deficient cells. This finding further supports a direct and indispensable ro