The phagocyte NADPH oxidase, dormant in resting cells, is activated during phagocytosis to produce superoxide, a precursor of microbicidal oxidants. The activated oxidase is a complex of membrane-integrated cytochrome bs58, composed of 91-kDa (gp91PbOx) and 22-kDa (p22PIox) subunits, and two cytosolic factors (p47PbOX and p67Pb°'), each containing two Src homology 3 (SH3) domains.Here we show that the region of the tandem SH3 domains of p47Phox (p47-SH3) expressed as a glutathione S-transferase fusion protein inhibits the superoxide production in a cell-free system, indicating involvement of the domains in the activation. Furthermore, we find that arachidonic acid and sodium dodecyl sulfate, activators of the oxidase in vitro, cause exposure of p47-SH3, which has probably been masked by the C-terminal region of this protein in a resting state. The unmasking of p47-SH3 appears to play a crucial role in the assembly of the oxidase components, because p47-SH3 binds to both p22PhO and p67PhOx but fails to interact with a mutant p22Phox carrying a Pro-156 -* Gln substitution in a prolinerich region, which has been found in a patient with chronic granulomatous disease. Based on the observations, we propose a signal-transducing mechanism whereby normally inaccessible SH3 domains become exposed upon activation to interact with their target proteins.During ingestion of microbes or upon stimulation with various soluble molecules, neutrophils and other phagocytic cells produce superoxide (O°), a precursor of microbicidal oxidants (1-4). The process involves activation ofthe phagocyte NADPH oxidase, dormant in resting cells, that catalyzes reduction of molecular oxygen to superoxide in conjunction with oxidation of NADPH. The significance of the NADPH oxidase in host defense is made evident by recurrent and life-threatening infections that occur in patients with chronic granulomatous disease (CGD) whose phagocytes lack the superoxide-producing system (1-4).The active NADPH oxidase is found on the phagocyte membrane as an enzyme complex, the components ofwhich are identified as targets of genetic defects causing CGD. The one identified at an earlier stage is a phagocyte-specific membrane-integrated b-type cytochrome, cytochrome b558 (5-11), composed of 91-kDa and 22-kDa subunits (designated gp91Phox and p22PhOX, respectively). The cytochrome is now considered to be a flavocytochrome comprising an apparatus transporting electrons from NADPH via FAD and then heme to molecular oxygen (12)(13)(14)(15)(16) . In addition to these specialized factors, as a third cytosolic factor, the small GTPbinding protein p21rac (rac 1 and/or rac 2) is also involved in the system (22)(23)(24).Although the components of the NADPH oxidase are thus identified, little is known about the mechanism for their assembly leading to activation of the enzyme. Upon phagocyte stimulation, the cytosolic components translocate to the membrane where cytochrome b558 resides (25,26). Experiments using neutrophils from CGD patients have revealed that the t...