Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b558, a heterodimer composed of a 22-kDa protein (p22phox) noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91phox). CGD phagocytes lack both p22phox and gp91phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91phox has not been reported. To investigate the stability and interaction of cytochrome b558 subunits, we introduced p22phox and gp91phox cDNA into recombinant baculoviruses. Recombinant gp91phox (rgp91phox) and p22phox (rp22phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22phox/rgp91phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b558 subunits in eukaryotic cells.