We hypothesized that xanthine oxidase (XO)-derived hydrogen peroxide (H2O2) contributes to ischemic skeletal muscle injury during reperfusion. We found that after ischemia (3 h) and then reperfusion (4 h) rat gastrocnemius muscles had decreased contractile function following direct stimulation. Three lines of investigation suggested that XO-derived H2O2 contributes to reperfusion injury of ischemic skeletal muscle. First, treatment with dimethylthiurea (DMTU), a highly permeant O2 metabolite scavenger, but not urea, just before reperfusion improved muscle function in legs subjected to ischemia and then reperfusion. Second, gastrocnemius muscles from rats fed tungsten or allopurinol had negligible XO activities and increased muscle function after ischemia and reperfusion. Third, as assessed by measurement of skeletal muscle catalase activity in the presence of aminotriazole, H2O2 was measured during reperfusion of ischemic muscles from untreated or urea-treated rats but not during reperfusion of muscles from rats treated with DMTU, tungsten, or allopurinol.