NADPH oxidase is an enzyme that converts molecular oxygen into reactive oxygen species, which cause severe damage in several organs. Cyclooxygenase (COX)-2 is an inducible enzyme that is important in gastric mucosal defense and repair processes. It is unclear whether NADPH oxidase is related to COX expression in the gastric mucosa, so we investigated the correlation. Under urethane anesthesia, a male Sprague Dawley rat stomach was mounted in an ex-vivo chamber, and ischemia/reperfusion (I/R) was performed through a cannula in the femoral vein. I/R significantly increased NADPH oxidase activity, H₂O₂ production, and myeloperoxidase (MPO) activity. In contrast, ischemia alone clearly enhanced both NADPH oxidase activity and H₂O₂ production but not MPO activity. Pretreatment with the NADPH oxidase inhibitor diphenylene iodonium (DPI) suppressed I/R-induced mucosal damage. On the other hand, the selective COX-2 inhibitor rofecoxib exhibited a tendency to enhance the severity of gastric damage induced by I/R, although the selective COX-1 inhibitor SC-560 and the nonselective COX inhibitor indomethacin had no effect. I/R also increased the expression of COX-2, and this increase was suppressed by pretreatment with DPI. These findings suggest that the increase in NADPH oxidase activity is involved in the occurrence of gastric mucosal damage induced by I/R and that this enzyme activity may be causally related to the upregulation of COX-2 during I/R.