While primary neuronal cell cultures have been used to investigate excitotoxicity, development of cell lines exhibiting glutamate receptor-mediated death is desirable. P19 mouse embryonal carcinoma cells, exposed to retinoic acid and plated onto a layer of cultured mouse cortical glial cells, differentiated into neuron-like elements immunoreactive for neurofilaments and neuron-specific enolase. Whole-cell recordings revealed inward currents in response to extracellular application of either NMDA or kainate. The NMDA-induced currents exhibited a voltage-dependent blockade by magnesium, required glycine for maximal activation, and were blocked by the NMDA antagonist dizocilpine. Kainate-induced currents were blocked by the AMPA/kainate receptor antagonist CNQX. Exposure to 500 μM NMDA for 24 h destroyed most P19 cells (EC50 approximately 70 μM); death was prevented by dizocilpine or D-APV. Exposure to 500 μM kainate also resulted in widespread death reduced by CNQX. Thus differentiated P19 cells exhibited both excitatory amino acid responses and vulnerability to excitotoxicity, characteristic of CNS neurons. These cells may provide a genetically open system useful for studying glutamate receptor-mediated phenomena at a molecular level. © 1993 John Wiley & Sons, Inc.