To study the role of calcium in neuronal death during ischemia, we examined the characteristics of intracellular Ca2+ ([Ca2+]i) changes in single rat forebrain neurons exposed for 5 min to glutamate (3 microM + 1 microM glycine), NMDA (30 microM + 1 microM glycine), kainate (100 microM) or high K+ (50 mM), under both normal and ischemic conditions. The parameters of [Ca2+]i change measured included peak [Ca2+]i level, plateau [Ca2+]i level, area under the [Ca2+]i response curve and time taken by [Ca2+]i to recover to 10% of the peak response. Under normal conditions, all the agonists studied produced similar [Ca2+]i changes. Chemical ischemia simulated by application of 5 mM KCN in glucose-free buffer had no effect on the basal level of [Ca2+]i, but significantly enhanced and prolonged the [Ca2+]i changes produced by all the agonists. However, in toxicity studies, chemical ischemia significantly potentiated the toxicity of only glutamate and N-methyl-D-aspartate. In correlation studies, all the neurons which died displayed an irreversible secondary [Ca2+]i load prior to loss of viability. These studies suggest that while Ca2+ entry may play a critical role in neuronal death, the magnitude of initial [Ca2+]i change does not predict the toxicity of an agonist in cortical neurons.