Platelets play an important role in atherosclerotic and thromboembolic vascular diseases. It has been reported that reactive oxygen species (ROS) could modify platelet function, and platelets themselves have the ability to produce ROS. However, the enzymatic sources of ROS in platelets have not been fully determined. The NADH/NADPH oxidase system was originally identified as the major source of ROS in phagocytes. Recently, it has become evident that this oxidase is functionally expressed not only in phagocytes but also in various cell types. The present study was undertaken to test the hypothesis that NADH/NADPH oxidase might be expressed in human platelets. Lucigenin-enhanced chemiluminescence (L-CL) and electron spin resonance (ESR) method demonstrated that human platelets obtained from healthy volunteers released ROS, and the released ROS were increased by stimulation with 12-O-tetradecanoylphorbol-13-acetate (TPA) or calcium ionophore. Homogenates of human platelets, as well as MEG01 cells, megakaryocytic cell line, had the enzymatic activity to produce superoxide in NADH/NADPH-dependent manners. This enzymatic activity was suppressed by diphenylene iodonium (DPI), an inhibitor of NADH/NADPH oxidase. Western blot analysis demonstrated that platelets and MEG01 cells expressed p22(phox) and p67(phox) proteins, components of NADH/NADPH oxidase. Thus, human platelets have the enzymatic activity of p22(phox)-based NADH/NADPH oxidase, and this oxidase is likely one of the important sources of ROS in platelets.