Heme oxygenase-1 (HO-1) has been demonstrated to protect against tissue injury. Furthermore, HO-1 is also shown to be antioxidant. Our recent findings indicate that acute alcohol (EtOH) intoxication exacerbates postburn intestinal and lung tissue damage, and this was found to be neutrophil dependent. Because neutrophil-mediated tissue injury involves the release of superoxide anions (O2-), the present study examined the role of HO-1 in neutrophil O2- production following EtOH and burn injury. Furthermore, we investigated whether HO-1 antioxidant properties are mediated via modulation of p47phox and/or p67phox proteins. Male rats (∼250 g) were gavaged with EtOH to achieve a blood EtOH level of ∼100 mg/dL before burn or sham injury (∼12.5% total body surface area). Some rats were treated with HO-1 activator cobalt protoporphyrin IX chloride (Copp; 25 mg/kg body weight) at the time of injury. On day 1 after injury, we found that EtOH combined with burn injury significantly increased neutrophil O2- production and p47phox and p67 phox activation and decreased caspase-3 activity and apoptosis. This was accompanied with a decrease in neutrophil HO-1 levels. The treatment of animals with HO-1 activator Copp normalized neutrophil HO-1, O2-, p47phox, and p67phox following EtOH and burn injury. The expression of caspase-3, however, was further decreased in Copp-treated sham and EtOH plus burn groups. Moreover, Copp treatment also prevented the increase in intestinal edema and permeability following EtOH and burn injury. Altogether, these findings provide a new insight into the mechanism by which HO-1 regulates neutrophil O2- production and protect the intestine from damage following EtOH and burn injury. Copyright © 2008 by The American Association of Immunologists, Inc.