The NADPH-dependent superoxide production induced by sodium dodecyl sulfate (SDS) in the sonicates of unstimulated pig neutrophils required both membrane fraction and two components of cytosol fraction. The potency of the cytosol fraction in the activation of the superoxide production could be reconstituted dose dependently by mixing two protein components with relative molecular masses of 300 kDa and 50 kDa. Another low-molecular-mass component (1.3 kDa) could substitute the 50-kDa component. In the cell-free system consisting of the 300- and 50-kDa components and the membrane fraction, the superoxide production was markedly enhanced by FAD with a required concentration for half-maximal effect of 0.16 microM and inhibited by divalent cations such as Ca2+, Ba2+, Co2+, Zn2+ and Mn2+ and not Mg2+. ATP was not necessary for the activation, indicating that protein kinases such as protein kinase C are not involved in the SDS-dependent activation of NADPH oxidase. The NADPH oxidase activated by SDS in the cell-free system was recovered in the membrane fraction, and the superoxide formation by the SDS-activated membrane exhibited a Km value for NADPH of 46 microM and optimum pH at 7.0. The formation did not require the addition of SDS and FAD to the reaction mixture and was scarcely inhibited by the divalent cations.