To examine the neurochemical effects of chronic hypoxia on immature nervous tissue in vitro, mixed neuronal–glial cell cultures derived from fetal mice were exposed to 5% O2 for 24 or 48 h. Those cultures subjected to longer hypoxia manifested improved neuronal survival compared to those with the shorter insult, both with respect to neuronal morphology and also cell counts. Neurochemical assays were performed on living cells in situ to determine the possible basis for differential cell survival. After both exposure conditions, Ro5–4864‐displaceable benzodiazepine (BDZ) binding, reflecting nonneuronal BDZ binding sites, was either not reduced or was elevated. Although initially reduced, binding of the excitatory amino acid (EAA) glutamate was progressively increased after both insults and, within 2 days after return to normoxia, was increased relative to control values (121 and 128% of controls, P < 0.05). The most impressive neurochemical differences between the two conditions related to changes in the predominantly or exclusively glial functions of glutamate uptake and glutamine synthetase activity. In those cultures with relatively preserved neuronal morphology: 1) high affinity uptake of glutamate was elevated compared to the shorter hypoxic insult by 3 days of recovery (104 vs 70%, P < 0.001) and 2) glutamine synthetase, an enzyme localized primarily within astrocytes, was significantly elevated even when compared to absolute control values (148%, P < 0.001). These data suggest that longer periods of hypoxia may be less deleterious to neurons than shorter hypoxic events because of a time‐dependent stimulation of specific glial cell functions which relate to increased metabolism of potentially neurotoxic EAAs such as glutamate.