The heterodimeric, integral membrane protein flavocytochrome b (Cyt b) is the catalytic core of the phagocyte NADPH oxidase and generates superoxide which plays a critical role in host defense. To better define the activation of superoxide production by this multisubunit enzyme complex, Cyt b-specific monoclonal antibodies (mAbs) and the p41phox SH3 domains (P47SH3 AB) were used in the present study as probes to map surface structure and conformational dynamics in human neutrophil Cyt b. In pull-down and co-immunoprecipitation studies with detergent-solubilized Cyt b, the oxidase-inhibitory mAb CS9 was shown to share an overlapping binding site with P47SH3AB on the C-terminal region of the p22phox subunit. Similar studies demonstrated a surprising lack of overlap between the mAb 44.1 and CS9/P47SH3AB binding sites, and they indicated that the oxidase-inhibitory mAb NL7 binds a region physically separated from the p22 phox C-terminal domain. Resonance energy transfer and size exclusion chromatography confirmed the above results for functionally reconstituted Cyt b and provided evidence that binding of both mAb CS9 and P47SH3AB altered the conformation of Cyt b. Further support that binding of the p47 phox SH3 domains modulates the structure of Cyt b was obtained using a cell-free assay system where P47SH3AB enhanced superoxide production in the presence of a p67phox (1 -212)-Rac1(Q61L) fusion protein. Taken together, this study further characterizes the structure of human neutrophil Cyt b in both detergent micelles and reconstituted membrane bilayers, and it provides evidence that the cytosolic regulatory subunit p47phox modulates the conformation of Cyt b (in addition to serving as an adapter protein) during oxidase activation.