Activation of the phagocyte NADPH oxidase complex requires assembly of the cytosolic factors p47PHOX, p67PHOX, p40PHOX, and Rac with the membrane-bound cytochrome b558. We recently established a direct interaction between p67PHOX and cytochrome b558. In the present study, we show that removal of the C-terminal domain of p67PHOX increased its binding to cytochrome b558. Whereas phosphorylated p40PHOX alone did not bind to cytochrome b558, phosphorylated p47PHOX did, and, moreover, it allowed the binding of p40PHOX to the cytochrome. Furthermore, both increased the binding of p67PHOX) to the cytochrome. Phosphorylated p47PHOX thus appears to increase the binding of p67(PHOX) to cytochrome b558 by serving as an adapter, bringing p67PHOX into proximity with cytochrome b558, whereas phosphorylated p40(PHOX) may increase the binding by inducing a conformational change that allows p67PHOX to interact fully with cytochrome b558.