The excitotoxic response of NT2-N cells, a clonal line of human teratocarcinoma cells that are terminally differentiated into neuron- like cells, was examined using several endpoints. A 15 min exposure to glutamate produced a dose-dependent toxicity with a maximal cell loss of 80–90% in 6 week old cells. The rapidly triggered excitotoxicity induced by glutamate was blocked by NMDA selective antagonists, was calcium dependent and pH sensitive and could be mimicked by NMDA but not by non-NMDA agonists, AMPA, kainate or quisqualate. The non-NMDA agonists however caused toxicity on prolonged exposure. The NMDA receptor modulators glycine and spermidine enhanced glutamate-mediated toxicity whereas ifenprodil potently and completely inhibited toxicity suggesting that the toxic response is mediated by the NR1/NR2B combination of NMDA subunits. These cells can be rescued from death up to 1 hr after removal of glutamate by NMDA receptor blockade, removal of extracellular Ca2+ or lowering of pH. The extent of rescue is directly related to the time elapsed before intervention. Blockage of NMDA receptor activity for 1 hr immediately after removal of glutamate is both necessary and sufficient for complete rescue. Glutamate- mediated toxicity was not prevented by nitric oxide synthase inhibitors nor was nitric oxide synthase detected in NT2-N cells indicating that nitric oxide is not required for glutamate-mediated excitotoxicity. In summary, NT2-N cells exhibit a robust excitotoxic response and represent a novel model system in which to study the molecular basis of excitotoxic cell death.