Oxygen free radicals have been implicated in the pathogenesis of cerebral ischemia and reperfusion injury. 4-Hydroxy-2,2,6,6-tetramethylpiperidene-1-oxyl (Tempol) has been reported as a stable nitroxide and a membrane-permeable free radical scavenger. This study was performed to investigate the mechanism of Tempol in attenuating ischemia–reperfusion injury in a rat model of transient focal cerebral ischemia. We measured the cerebral 2,3-dihydroxybenzoic acid (DHBA) level as the amount of hydroxyl radical production using a microdialysis technique with salicylic acids trapping during ischemia and reperfusion. The concentration of cerebral thiobarbituric acid reactive substances (TBARS), representing the extent of lipid peroxidation by free radicals, and the area of cerebral infarction were also measured. The level of cerebral 2,3-DHBA was increased during ischemia and reperfusion, especially during the early reperfusion stage at the periphery of the infarct area (nearly 500-fold). Intravenous administration of Tempol at the time of reperfusion reduced 2,3-DHBA production (Vehicle group: 472.2±196.2, Tempol group: 238.3±77.2) and the cerebral TBARS level (Vehicle group: 541.7±84.7, Tempol group: 339.0±147.2 nmol/g), and decreased the size of the cerebral infarction (Vehicle group: 202.2±98.4, Tempol group: 98.5±13.7 mm³). In contrast, Tempol administered 15 min prior to reperfusion reduced neither the TBARS level nor the size of the infarction. These results indicate that Tempol administration at the time of reperfusion reduced lipid peroxidation by scavenging free radicals, resulting in a reduction of the infarct size.