In a previous paper, we described the kinetic characteristics of the inhibition exerted by the protease inhibitors tosylphenylalanyl and tosyllysyl chloromethanes on superoxide production by human polymorphonuclear leukocytes when stimulated by phorbol esters [E. C. Conseiller & F. Lederer (1989) Eur. J. Biochem. 183, 107-1141. The results suggested the existence of a specific target which was affinity labeled by the inhibitors. The target appeared to be neither a protease, nor intracellular enzymes which can be inhibited in vitro by the chloromethanes (protein kinase C, hexokinase and enzymes of the hexose monophosphate shunt). In the present work, using the cell-free reconstitution assay for superoxide production, we substantiate the hypothesis that the chloromethanes, target is on the plasma membrane. We have radiolabeled the membranes of cells inactivated before or after phorbol ester stimulation, using either [3H]KBH4 reduction after reaction with unlabeled inactivator, or tritiated tosylphenylalanyl chloromethane. In all cases, besides a certain background of non-specific labeling, a radioactive band of M , 15 000 can be observed upon SDSjPAGE of radiolabeled membranes. We suggest that it is the chemical modification of this protein which is responsible for inactivation of superoxide production. Its identity and its role in the oxidative burst remain to be determined.NADPH oxidase, an enzymatic complex present in blood phagocytes, produces superoxide anion when the cells are stimulated by soluble or particulate stimuli. This oxygen derivative then undergoes a series of enzymatic and non-enzymatic reactions producing a variety of oxidizing agents which play an essential role in host defense mechanisms against infectious diseases [I -41. Various studies have demonstrated a requirement for at least two redox components, a flavoprotein [5 -71 and a low-potential cytochrome b termed cytochrome b245 or b55t,, which is partially or totally absent from patients with X-linked chronic granulomatous disease (CGD) [8 -101. In activated cells, the superoxide anion-producing activity is found attached to the plasma membrane [I1 -131.Proteins other than the redox components are required for appearance of the enzymatic activity, which is dormant in resting cells. Studies initiated by Bromberg and Pick [14] and Curnutte [I51 have shown it is possible to induce the oxidase activity in a cell-free system by combining the cytosolic and particulate fractions of resting cells in the presence of SDS or arachidonate. Studies with such a system led to the implication Abbreviations. CGD, chronic granulomatous disease; Dip-F, diisopropyl fluorophosphate; HBSS; Hank's balanced salt solution; Me'SO; dimethylsulfoxide; PhMeS02F, phenylmethanesulfonyl fluoride; PMA, phorbol12-myristate 13-acetate; PMN, polymorphonuclear leukocytes ; TosLysCH2C1,N"-p-tosyl-L-lysyl chloromethane; TosPheCH2C1, N-p-tosyl-1,-phenylalanyl chloromethane.Enzymes. NADPH oxidase (EC 1.6.99.6); superoxide dismutase (EC 1.25.1.1) ~-of several cytosolic factors in...