Generation of superoxide (O2-) by the NADPH-dependent oxidase of polymorphonuclear leukocytes is an essential component of the innate immune response to invading microorganisms. To examine NADPH oxidase function during phagocytosis, we evaluated its activation and assembly following ingestion of serum-opsonized Neisseria meningitidis, serogroup B (NMB), and compared it with that elicited by serum-opsonized zymosan (OPZ). Opsonized N. meningitidis- and OPZ-dependent generation of reactive oxygen species by polymorphonuclear leukocytes peaked early and then terminated. Phosphorylation of p47phox coincided with peak generation of reactive oxygen species by either stimulus, consistent with a role for p47phox phosphorylation during NADPH oxidase activation, and correlated with phagosomal colocalization of flavocytochrome b558 (flavocytochrome b) and p47phox and p67phox (p47/67phox). Termination of respiratory burst activity did not reflect dephosphorylation of plasma membrane- and/or phagosome-associated p47phox; in contrast, the specific activity of phosphorylated p47phox at the phagosomal membrane increased. Most significantly, termination of oxidase activity paralleled the loss of p47/67phox from both NMB and OPZ phagosomes despite the continued presence of flavocytochrome b. These data suggest that 1) the onset of respiratory burst activity during phagocytosis is linked to the phosphorylation of p47phox and its translocation to the phagosome; and 2) termination of oxidase activity correlates with loss of p47/67phox from flavocytochrome b-enriched phagosomes and additional phosphorylation of membrane-associated p47phox.