The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b558 and four cytosolic proteins; p67phox, p47phox, p40phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b558. However, a number of studies have suggested the presence of another site on the p67phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2',3'-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex.