Pretreatment with 3-nitropropionic acid (3-NPA) has been shown to induce tolerance to ischemic/hypoxic brain damage. However, regional differences in tolerance induction by 3-NPA and the degree to which impaired glucose metabolism due to 3-NPA pretreatment itself is directly involved remain unknown. To evaluate these issues using dynamic positron autoradiography with [(18)F]2-fluoro-2-deoxy-D-glucose, the cerebral glucose metabolic rate (CMRglc) was serially measured before and after hypoxia-loading in rat brain slices pretreated with 3-NPA. CMRglc before hypoxia did not significantly differ between the 3-NPA pretreatment group and control group. The 3-NPA-associated recovery of CMRglc after reoxygenation was observed in the frontal cortex, hippocampus, and cerebellum, but not in the striatum and thalamus. Thus, we demonstrated the induction of region-specific hypoxic tolerance after 3-NPA pretreatment using CMRglc maintenance as an index of neuronal viability, and it is unlikely that this induction is associated with the persistence of impaired glucose metabolism due to 3-NPA pretreatment.