BACKGROUND & AIMS Superoxide anion (O2-) plays an important role in gastric pathophysiology. The aims of this study were to identify O2--producing activity in gastric mucosal cells and to elucidate its possible roles in inflammatory responses of the cells. METHODS The amount of O2- was measured by the reduction of cytochrome c, and O2--producing cells were visualized by nitroblue tetrazolium reaction. Cytosolic components of the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase were detected by immunoblotting and immunocytochemical analyses with antibodies against p47-phox and p67-phox. RESULTS Gastric pit cells, but not parietal cells, spontaneously released O2- at 50 nmol . mg protein-1 . h-1. NADPH or guanosine 5'-O-(3-thiotriphosphate) increased the release more than threefold, whereas diphenylene iodonium inhibited it. A reconstituted cell-free system showed that both membrane fraction and neutrophil-related cytosolic components were required for the activity. p47-phox and p67-phox were expressed in the cells. Live Helicobacter pylori organisms and their culture supernatants significantly increased the O2- release. Furthermore, H. pylori lipopolysaccharide could enhance the release more effectively than Escherichia coli lipopolysaccharide. The O2--dependent activation of nuclear factor kappaB occurred in these primed cells. CONCLUSIONS Gastric pit cells may actively regulate inflammatory responses of gastric mucosa through a phagocyte NADPH oxidase-like activity.