The assembly of cytosolic p47 phox and p67 phox with flavocytochrome b 558 at the membrane is crucial for activating the leukocyte NADPH oxidase that generates superoxide for microbial killing. p47 phox and p67 phox are linked via a high affinity, tail-to-tail interaction involving a proline-rich region (PRR) and a C-terminal SH3 domain (SH3b), respectively, in their C-termini. This interaction mediates p67 phox translocation in neutrophils, but is not required for oxidase activity in model systems. Here we examined phagocytosis-induced NADPH oxidase assembly, showing the sequential recruitment of YFP-tagged p67 phox to the phagosomal cup, and, after phagosome internalization, a probe for PI(3)P followed by a YFP-tagged fragment derived from the p47 phox PRR. This fragment was recruited in a flavocytochrome b 558-dependent, p67 phox-specific, and PI(3)P-independent manner. These findings indicate that p47PRR fragment probes the status of the p67 phox SH3b domain and suggest that the p47 phox/p67 phox tail-to-tail interaction is disrupted after oxidase assembly such that the p67 phox-SH3b domain becomes accessible. Superoxide generation was sustained within phagosomes, indicating that this change does not correlate with loss of enzyme activity. This study defines a sequence of events during phagocytosis-induced NADPH oxidase assembly and provides experimental evidence that intermolecular interactions within this complex are dynamic and modulated after assembly on phagosomes.