The human neutrophil NADPH oxidase is a multi-component complex composed of membrane-bound and cytosolic proteins. During activation, cytosolic proteins p47(phox), p67(phox), Rac2, and possibly p40(phox) translocate to the plasma membrane and associate with flavocytochrome b to form the active superoxide-generating system. To further investigate the role of p67(phox) in this complex assembly process, experiments were performed to identify possible regions of interaction between p67(phox) and other NADPH oxidase proteins. Using random sequence peptide phage-display library analysis of p67(phox), we identified a novel region in p47(phox) encompassing residues 323-332 and a previously identified SH3 binding domain encompassing p47(phox) residues 361-370 as p67(phox) binding sites. Synthetic peptides mimicking p47(phox) residues 323-332 inhibited the p47(phox)-p67(phox) binding interaction in an affinity binding assay; however, peptides mimicking flanking regions were inactive. Surprisingly, this same region of p47(phox) was found previously to represent a site of binding interaction for flavocytochrome b (DeLeo, F. R., Nauseef, W. M., Jesaitis, A. J., Burritt, J. B., Clark, R. A., and Quinn, M. T.(1995) J. Biol. Chem. 270, 26246-26251), and this observation was confirmed in the present report using two different in vitro assays that were not evaluated previously. Using affinity binding assays, we also found that p67(phox) and flavocytochrome b competed for binding to p47(phox)after activation, suggesting that prior to full NADPH oxidase assembly the 323-332 region of p47(phox) is associated with p67(phox) and at some point in the activation process is transferred to flavocytochrome b. Thus, taken together our data demonstrate that both p67(phox) and flavocytochrome b utilize a common binding site in p47(phox), presumably at distinct stages during the activation process, and this p47(phox) region plays a key role in regulating NADPH oxidase assembly.