We studied the effect of hyperbaric oxygen (HBO) preconditioning on the molecular mechanisms of neuroprotection in a rat focal cerebral ischemic model. Seventy two male Sprague-Dawley rats were pretreated with HBO (100% O₂, 2 atmospheres absolute, 1 hour once every other day for five sessions) or with room air. In experiment 1, HBO preconditioned rats and matched room air controls were subjected to focal cerebral ischemia or sham surgery. Post-injury motor parameters and infarction volumes of HBO preconditioned rats were compared with those of controls. In experiment 2, HBO preconditioned rats and matched room air controls were killed at different time points. Brain levels of hypoxia-inducible factor 1 (HIF-1) and its downstream target gene erythropoietin (EPO) analyzed by Western blotting and RT-PCR as well as HIF-1 DNA-binding and transcriptional activities were determined in the ipsilateral hemisphere. HBO induced a marked increase in the protein expressions of HIF-1 and EPO and the activity of HIF-1, as well as the expression of EPO mRNA. HBO preconditioning dramatically improved the neurobehavioral outcome at all time points (3.0±2.1 vs 5.6±1.5 at 4 h, 5.0±1.8 vs 8.8±1.4 at 8 h, 6.4±1.8 vs 9.7±1.3, P<0.01 respectively) and reduced infarction volumes (20.7±4.5% vs 12.5±3.6%, TTC staining) after cerebral ischemia. This observation indicates that the neuroprotection induced by HBO-preconditioning may be mediated by an upregulation of HIF-1 and its target gene EPO.