Background and Purpose-The role of mitochondrial ATP-sensitive potassium channels (mitoK ATP ) in ischemic tolerance has been well documented in heart, but little work has been done in brain. To investigate the involvement of mitoK ATP activation in chemical preconditioning in brain, we examined the effect of 5-hydroxydecanoate (5-HD), a selective mitoK ATP blocker, on neurotoxin 3-nitropropionic acid (3-NPA)-induced ischemic tolerance to transient focal cerebral ischemia in rats. Methods-Male Wistar rats were administrated 3-NPA (20 mg/kg IP; nϭ16) or vehicle (saline; nϭ16) 3 days before temporary occlusion (120 minutes) of the middle cerebral artery; 5-HD (40 mg/kg IP; nϭ16) was injected 20 minutes before 3-NPA administration. Infarct volumes were measured 4 days after reperfusion. To directly investigate whether chemical preconditioning activates mitoK ATP , we tested the effect of prior incubation with 1 mmol/L 5-HD on 300 mol/L 3-NPA-induced alterations of mitochondrial membrane potential (⌬⌿ m ) in cultured neurons and astrocytes using the fluorescent dye tetramethylrhodamine ethyl ester. Results-Treatment with 3-NPA exhibited a 16% reduction (PϽ0.05) and 23% reduction in infarct volume (PϽ0.01) for total brain and cortex, respectively. Pretreatment with 5-HD completely abolished the neuroprotective effect of chemical preconditioning. In cultured cells, 3-NPA resulted in mitochondrial depolarization. This change of ⌬⌿ m was completely blocked by 5-HD pretreatment. Conclusions-These results strongly suggest that opening of mitoK ATP plays a key role as the trigger in the development of 3-NPA-induced ischemic tolerance in brain.