Human polymorphonuclear neutrophils (PMNs) possess a potent oxygen- dependent microbicidal system that depends on the activity of a stimulus-activated multicomponent nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Patients with chronic granulomatous disease (CGD) lack activity of this oxidase and consequently suffer severe and frequent infections. Components of the oxidase include both membrane- bound factors (most notably, cytochrome b559, which is absent in the X- linked form of CGD) and at least two cytosolic factors, one or the other of which is absent in autosomal CGD. Patients with CGD, particularly the autosomal type, have defective phosphorylation of proteins in the 44 to 48 Kd range. A polyclonal antiserum (B-1) that recognizes cytosolic oxidase components of 47 and 67 Kd was used to identify phosphoproteins in a cell-free oxidase system. Two-dimensional gel electrophoresis showed the identity of the 47-Kd cytosolic protein (p47-phox) recognized by B-1 and the cationic 47-Kd protein that is phosphorylated in normal but not p47-phox-deficient CGD cytosol during activation of the NADPH-dependent oxidase. All full-length and C- terminal recombinant p47-phox proteins augmented the superoxide- generating capacity of the cell-free system and were phosphorylated when added to cytosol from normal subjects or from a patient with p47- deficient autosomal CGD. These studies provide compelling evidence that the 47-Kd cationic protein that is a substrate for phosphorylation during the activation of PMNs is, in fact, p47-phox, a cytosolic protein previously shown to be critical for normal activity of the NADPH-dependent oxidase of PMNs.