Exhaustive exercise generates free radicals. However, the source of this oxidative damage remains controversial. The aim of this paper was to study further the mechanism of exercise-induced production of free radicals. Testing the hypothesis that xanthine oxidase contributes to the production of free radicals during exercise, we found not only that exercise caused an increase in blood xanthine oxidase activity in rats but also that inhibiting xanthine oxidase with allopurinol prevented exercise-induced oxidation of glutathione in both rats and in humans. Furthermore, inhibiting xanthine oxidase prevented the increases in the plasma activity of cytosolic enzymes (lactate dehydrogenase, aspartate aminotransferase, and creatine kinase) seen after exhaustive exercise. Our results provide evidence that xanthine oxidase is responsible for the free radical production and tissue damage during exhaustive exercise. These findings also suggest that mitochondria play a minor role as a source of free radicals during exhaustive physical exercise.