In animal models of severe ischemia, it has not been uniformly observed that anesthetics are protective. However, anesthetics have not been evaluated in the presence of a mild excitotoxic insult. We hypothesized that in the presence of a mild excitotoxic insult, 3 microm N-methyl-D-aspartate (NMDA), isoflurane may prevent apoptotic cell death. Primary mixed neuronal/glial cultures were prepared from fetal rat brains. Mature cultures were exposed to dissolved isoflurane [0 mM, 0.4 mM (1.8 minimum alveolar concentration) or 1.6 mM (7 minimum alveolar concentration)] or dizocilpine (10 microM), and NMDA (0 or 3 microM) at 37 degrees C for 30 minutes. Apoptosis was assessed using terminal-deoxy-nucleotidyl end-nick labeling oligonucleosomal DNA fragmentation enzyme-linked immunosorbent assay, and caspases-3 and -9 activation assays. NMDA (3 muM) induced apoptosis in mixed neuronal/glial cell cultures. Apoptosis induced by 3 microm NMDA was caspase-3 but not caspase-9 mediated. In the presence of a mild excitotoxic insult, this investigation showed an attenuation of apoptotic cell death by dizocilpine, but not isoflurane.