Cultures of rat hippocampal pyramidal neurons were used to examine the roles of excitatory synaptic transmission, NMDA receptors, and elevated [Ca2+]i in the production of excitotoxicity. In integral of 70% of the cells observed, perfusion with Mg2(+)-free, glycine-supplemented medium induced large spontaneous fluctuations or maintained plateaus of [Ca2+]i. [Ca2+]i fluctuations could be blocked by tetrodotoxin, NMDA receptor antagonists, dihydropyridines, or compounds that inhibit synaptic transmission in the hippocampus, but not by the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione. When cells were treated with Mg2(+)-free, glycine-supplemented medium and examined 24 hr later, integral of 30% of the neurons were found to have died. Cell death could be inhibited by the same agents that reduced [Ca2+]i fluctuations. These results support a role for direct excitatory synaptic transmission, as opposed to the general release of glutamate, in excitotoxicity. A major role for synaptically activated NMDA receptors, rather than kainate/quisqualate receptors, is also indicated. Neuronal death may be produced by abnormal changes in neuronal [Ca2+]i.