Superoxide produced by the phagocyte reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is essential for host defense. Enzyme activation requires translocation of p67(phox), p47(phox), and Rac-GTP to flavocytochrome b558 in phagocyte membranes. To examine the regulation of phagocytosis-induced superoxide production, flavocytochrome b558, p47(phox), p67(phox), and the FcgammaIIA receptor were expressed from stable transgenes in COS7 cells. The resulting COS(phox)FcgammaR cells produce high levels of superoxide when stimulated with phorbol ester and efficiently ingest immunoglobulin (Ig)G-coated erythrocytes, but phagocytosis did not activate the NADPH oxidase. COS7 cells lack p40(phox), whose role in the NADPH oxidase is poorly understood. p40(phox) contains SH3 and phagocyte oxidase and Bem1p (PB1) domains that can mediate binding to p47(phox) and p67(phox), respectively, along with a PX domain that binds to phosphatidylinositol-3-phosphate (PI(3)P), which is generated in phagosomal membranes. Expression of p40(phox) was sufficient to activate superoxide production in COS(phox)FcgammaR phagosomes. FcgammaIIA-stimulated NADPH oxidase activity was abrogated by point mutations in p40(phox) that disrupt PI(3)P binding, or by simultaneous mutations in the SH3 and PB1 domains. Consistent with an essential role for PI(3)P in regulating the oxidase complex, phagosome NADPH oxidase activation in primary macrophages ingesting IgG-coated beads was inhibited by phosphatidylinositol 3 kinase inhibitors to a much greater extent than phagocytosis itself. Hence, this study identifies a role for p40(phox) and PI(3)P in coupling FcgammaR-mediated phagocytosis to activation of the NADPH oxidase.