The role of xanthine dehydrogenase and oxidase as a source of free radicals contributing to focal cerebral ischemic injury was evaluated in Long-Evans rats after the middle cerebral artery was permanently occluded and both carotid arteries were clamped for 90 min. The fraction of xanthine dehydrogenase present as the free radical producing oxidase increased slightly from 22% in control cortex to 30% in the ischemic right cortex during the first 3 h of reperfusion and then remained relatively unchanged over the next 24 h. This increase may in part be due to entrapped plasma, which contained 4.5 ± 0.8 nmol·min-1·ml-1 xanthine oxidase entirely in the free radical-producing form. Infarct volume was unaffected by pretreatment with 50 mg allopurinol/kg per day over 3 days before surgery but was decreased by 8% with 100 mg/kg and 24% with 150 mg/kg of allopurinol (P < 0.05). However, inhibition of xanthine oxidase by dietary depletion of the essential molybdenum cofactor increased infarct volume by 19%, suggesting that protection by allopurinol at higher dosages was independent of xanthine oxidase inhibition. Neither xanthine oxidase present in rat brain nor circulating in plasma appears to be the primary source of oxygen radicals that contributes to infarction in focal cerebral ischemia.