Oxygen-derived free radicals, particularly superoxide anion, are considered important mediators of intestinal injury induced by ischemia/reperfusion based on the protective effects of superoxide dismutase and allopurinol. A role for free radicals was investigated in a model of necrotizing enterocolitis (NEC) which was initiated by a luminal, as opposed to a vascular, insult. Intestinal loops of weanling rabbits received either saline (control loops) or a solution of 10 mg/ml casein and 50 mg/ml calcium gluconate acidified to pH 4 with proprionic acid (treated loops). When the animals were sacrificed 3 hours later, severe damage was noted in the treated loops, which included blunting of villi and edema, with all animals surviving. At 16 hours only 5 of 8 rabbits survived, and 3 had hemorrhagic necrosis. Control loops were normal in each case. Intravenous infusion of superoxide dismutase (4 mg/kg/hr), commencing 15 minutes after NEC induction, totally prevented intestinal injury. On the other hand, pretreatment with allopurinol, an inhibitor of xanthine oxidase, for 2 days (30 and 60 mg/kg by mouth) was not protective against intestinal damage. A cellular infiltration in treated loops was not histologically evident in the majority of animals at 3 hours after treatment, a finding confirmed by the minimal accumulation of 111In-labeled leukocytes in damaged and intact intestinal tissue. These results suggest that superoxide generated locally from sources other than xanthine oxidase play a critical and early role in experimental NEC and that superoxide dismutase may prove to be an effective therapy in this devastating neonatal disease.