The assembly of cytosolic subunits p47phox, p67phox, and p40phox with flavocytochrome b558 at the membrane is required for activating the neutrophil NADPH oxidase that generates superoxide for microbial killing. The p47phox subunit plays a critical role in oxidase assembly. Recent studies showed that the p47phox Phox homology (PX) domain mediates phosphoinositide binding in vitro and regulates phorbol ester-induced NADPH oxidase activity in a K562 myeloid cell model. Because the importance of the p47phox PX domain in neutrophils is unclear, we investigated its role using p47phox knock-out (KO) mouse neutrophils to express human p47phox and derivatives harboring R90A mutations in the PX domain that result in loss of phosphoinositide binding. Human p47phox proteins were expressed at levels similar to endogenous murine p47phox, with the exception of a chronic granulomatous disease-associated R42Q mutant that was poorly expressed, and wild type human p47phox rescued p47phox KO mouse neutrophil NADPH oxidase activity. Plasma membrane NAPDH oxidase activity was reduced in neutrophils expressing p47phox with Arg90 substitutions, with substantial effects on responses to either phorbol ester or formyl-Met-Leu-Phe and more modest effects to particulate stimuli. In contrast, p47phox Arg90 mutants supported normal levels of intracellular NADPH oxidase activity during phagocytosis of a variety of particles and were recruited to phagosome membranes. This study defines a differential and agonist-dependent role of the p47phox PX domain for neutrophil NADPH oxidase activation.