The present study was undertaken to investigate the comparative effects of rapid vs graded correction of chronic hypoxia in vitro. Cerebral cortical cell cultures obtained from fetal mice were exposed to 5% O2 for 24 h and returned immediately to room air for the following 24 h (Group I); comparable cultures were exposed to 5% O2 for 24 h followed by 10% O2 for an additional 24 h before return to room air (Group II). At the conclusion of the experimental protocol (time 0), partial pressure of oxygen in the bathing medium of Group I cultures was significantly higher than that of Group II and non-hypoxic controls (151 mmHg vs 124 and 132 mmHg, respectively; P < 0.05). Throughout the recovery period, Group II cultures evidenced improved neuronal survival (e.g. 35,800 vs 17,700 neurons/culture well at time 0, P < 0.01), decreased lactate dehydrogenase efflux into the bathing medium, relative preservation of neuronal morphology, as well as higher specific and clonazepam-displaceable benzodiazepine binding and GABA uptake. Glutamate binding was not differentially affected and glutamine synthetase activity, a predominantly glial marker, was only modestly increased after graded reoxygenation. These results demonstrate that gradual reoxygenation after prolonged hypoxia in vitro (i) improves neuronal survival compared to rapid reoxygenation and (ii) delays the manifestations of metabolic dysfunction even though the length of hypoxic exposure is increased. The findings are also consistent with the concept that a period of relative hyperoxia may contribute to hypoxia-induced neuronal injury.