Peroxynitrite (ONOO-) exhibits potent neurotoxicity and plays an important role in neuronal death, but no evidence shows that it is formed in the brain during ischemia or subsequent reperfusion. To detect the formation of ONOO-, we used a hydrolysis/HPLC procedure to measure the formation of 3-nitro-L-tyrosine (NO2-Tyr), which is considered to reflect attack of ONOO- on L-tyrosine residues of cellular components in the brain. Focal ischemia was produced by occluding the right common carotid and right middle cerebral arteries for 2 hours, and the ischemic area was reperfused by reopening the middle cerebral artery. After 2 hours of ischemia, the values of the ratio of NO2-Tyr to L-tyrosine were 0% +/- 0%, 0.42% +/- 0.13% and 0.29% +/- 0.10% in the noninfarct, periinfarct, and core-of-infarct regions, respectively. After 3 hours of reperfusion following 2 hours of ischemia, the ratio in the periinfarct region reached 0.89 +/- 0.22%, which was significantly higher than that in the core-of-infarct region (0.35 +/- 0.09%). The NO2-Tyr was not detected in 50 mg/kg of N-monomethyl-L-arginine-treated or sham-operated rats. Regional CBF in the periinfarct region decreased to 30.8 +/- 15.9 mL/100 g/min during occlusion, but recovered more rapidly than did that in the core-of-infarct region.