NADPH oxidase produces a large amount of reactive oxygen species (ROS) mainly in phagocytic cells. ROS are involved in NF-kappaB activation, cytokine expression and thus, pathogenesis of pancreatitis. However, the source of ROS in pancreatic acinar cells has not been clarified. Cerulein rapidly induces acute and edematous form of pancreatitis. We investigated whether pancreatic acinar cells contain NADPH oxidase, and whether NADPH oxidase mediates interleukin-6 (IL-6) in pancreatic acinar AR42J cells stimulated with cerulein. Expression of NADPH oxidase subunits and NADPH oxidase activity were determined in the cells by immunofluorescence staining and lucigenin luminescence, respectively. Oxidant-sensitive nuclear transcription factor NF-kappaB activation was monitored by electrophoretic mobility shift assay. IL-6 expression was determined by reverse transcription-polymerase chain reaction and enzyme-linked immunosorbant assay. NADPH oxidase inhibitor diphenylene iodonium (DPI), antioxidant rebamipide, and antisense oligonucleotides (AS ODNs) for NADPH oxidase subunits p22phox and p47phox were used to determine the involvement of NADPH oxidase in NF-kappaB activation and IL-6 expression in AR42J cells. As a result, pancreatic acinar AR42J cells constitutively express NADPH oxidase subunits p67phox and p47phox in the cytosol and Nox1 and p22phox in the membrane. Cerulein-stimulated NADPH oxidase activity and induced NF-kappaB activation and IL-6 expression in AR42J cells. Treatment of DPI or rebamipide and transfection of AS ODNs for NADPH oxidase subunits suppressed cerulein-induced NF-kappaB activation and IL-6 expression compared to S ODNs. In conclusion, NADPH oxidase may mediate the expression of inflammatory cytokines by stimulating NF-kappaB activation in pancreatic acinar cells during the course of pancreatitis.