The phagocyte NADPH oxidase generates superoxide for microbial killing, and includes a membrane-bound flavocytochrome b(558) and cytosolic p67(phox), p47(phox), and p40(phox) subunits that undergo membrane translocation upon cellular activation. The function of p40(phox), which binds p67(phox) in resting cells, is incompletely understood. Recent studies showed that phagocytosis-induced superoxide production is stimulated by p40(phox) and its binding to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide enriched in membranes of internalized phagosomes. To better define the role of p40(phox) in FcgammaR-induced oxidase activation, we used immunofluorescence and real-time imaging of FcgammaR-induced phagocytosis. YFP-tagged p67(phox) and p40(phox) translocated to granulocyte phagosomes before phagosome internalization and accumulation of a probe for PI3P. p67(phox) and p47(phox) accumulation on nascent and internalized phagosomes did not require p40(phox) or PI3 kinase activity, although superoxide production before and after phagosome sealing was decreased by mutation of the p40(phox) PI3P-binding domain or wortmannin. Translocation of p40(phox) to nascent phagosomes required binding to p67(phox) but not PI3P, although the loss of PI3P binding reduced p40(phox) retention after phagosome internalization. We conclude that p40(phox) functions primarily to regulate FcgammaR-induced NADPH oxidase activity rather than assembly, and stimulates superoxide production via a PI3P signal that increases after phagosome internalization.