The NADPH-dependent oxidase of human neutrophils is a multicomponent system including cytosolic and membrane proteins. Activation requires translocation of cytosolic proteins p47phox, p67phox, and Rac2 to the plasma membrane and association with the membrane flavocytochrome b to assemble a functioning oxidase. We report the location of a region in p47phox that mediates its interaction with flavocytochrome b. From a random peptide phage display library, we used biopanning with purified flavocytochrome b to select phage peptides that mimicked potential p47phox binding residues. Using this approach, we identified a region of p47phox from residue 323 to 342 as a site of interaction with flavocytochrome b. Synthetic peptides 315SRKRLSQDAYRRNS328, 323AYRRNSVRFL332, and 334QRRRQARPGPQSPG347 inhibited superoxide (O2-.) production in the broken cell system with IC50 of 18, 57, and 15 microM, respectively. 323AYRRNSVRFL332 and its derivative peptides inhibited phosphorylation of p47phox. However, the functional importance of this peptide was independent of its effects on phosphorylation, since 323AYRRNAVRFL332 inhibited O2-. production, but had no effect on phosphorylation. None of the peptides blocked O2-. production when added after enzyme activation, suggesting that they inhibited the assembly, rather than the activity, of the oxidase. Furthermore these peptides inhibited membrane association of p47phox in the broken cell translocation assay and O2-. production by electropermeabilized neutrophils, thereby supporting the interpretation that this region of p47phox interacts with flavocytochrome b.