The phagocyte NADPH oxidase flavocytochrome b558 is a membrane-bound heterodimer comprised of a glycosylated subunit, gp91(phox), and a nonglycosylated subunit, p22(phox). It contains two nonidentical heme groups that mediate the final steps of electron transfer to molecular oxygen (O2), resulting in the generation of superoxide ion (O2-). However, the location of the hemes within the flavocytochrome heterodimer remains controversial. In this study, we have used transgenic COS7 cell lines expressing gp91(phox), p22(phox), or both polypeptides to examine the relative role of each flavocytochrome b558 subunit in heme binding and O2- formation. A similar membrane localization was observed when gp91(phox) and p22(phox) were either expressed individually or coexpressed, as analyzed by confocal microscopy and immunoblotting of subcellular fractions. Spectral analysis of membranes prepared from COS7 cell lines expressing either gp91(phox) or both gp91(phox) and p22(phox) showed a b-type cytochrome with spectral characteristics identical to those of human neutrophil flavocytochrome b558. In contrast, no heme spectrum was detected in wild-type COS7 membranes or those containing only p22(phox). Furthermore, redox titration studies suggested that two heme groups were contained in gp91(phox) expressed in COS7 membranes, with midpoint potentials of -264 and -233 mV that were very similar to those obtained for neutrophil flavocytochrome b558. These results provide strong support for the hypothesis that gp91(phox) is the sole heme binding subunit of flavocytochrome b558. However, coexpression of gp91(phox) and p22(phox) in COS7 membranes was required to support O2- production in combination with neutrophil cytosol, indicating that the functional assembly of the active NADPH oxidase complex requires both subunits of flavocytochrome b558.