To assess the influence of brain immaturity on the participation of excitotoxicity in hypoxia-related cell damage, the effects of a 6h-exposure to toxic concentration (100 μM) of glutamate were studied in cultured neurons taken at two maturational stages, i.e. 6 and 13 days in vitro. Neuronal cells were isolated from the fetal rat forebrain and cultured in controlled chemically-defined medium with 95% air-5% CO2. As a function of time after exposure to glutamate, neurons were examined for their morphology, viability measured by the MTT procedure, energy metabolism reflected by 2-D-[3H]deoxyglucose specific uptake, and rates of protein synthesis assessed by [3H]leucine incorporation. Apoptosis and necrosis were detected in cell nuclei by a fluorescent dye, DAPI (4,6-diamidino-2-phenylindole). Data are expressed as percentage of changes as compared to controls maintained under standard conditions. In 6-day-old neurons, exposure to glutamate did not affect cell morphology or functional properties, the cell viability only decreasing not significantly by 7.2% (n=16 dishes) within 72h post-exposure. By contrast, a 6h-hypoxia (95% N2-5% CO2) followed by reoxygenation led to transient cell hypermetabolism (37.5%, n=46, p<0.01) 24h later, and to a cycloheximide-sensitive biphasic increase in protein synthesis 1h after the onset of the insult and 48h after reoxygenation (77% and 72%, respectively, n=26, p<0.01). Furthermore, 72h after reoxygenation, cell viability was decreased by 20% (n=30,p<0.01), while 20% of the neurons exhibited characteristic features of apoptosis. In 13-day-old neurons, exposure to glutamate reduced energy metabolism by 26% (p<0.01) as soon as the end of exposure(6h). Cell viability then decreased as a function of time to reach 27% below controls at 72h post-exposure. However, glutamate did not increase protein synthesis at any time, and the final percentage of apoptotic neurons (5.5%) was not different from controls (4.8%), whereas the number of necrotic neurons was higher (24% versus 5%). Therefore, immature neurons (6 days in vitro) are sensitive to transient hypoxia that induces a delayed, mainly apoptotic cell death, but are rather resistant to glutamate toxicity. More mature neurons (13 days) are sensitive to glutamate, but the excitatory amino acid effects do not correlate with those of hypoxia, and leads to unprogrammed, mainly necrotic cell death.