NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).