The flavocytochrome b558 of the phagocyte NADPH oxidase complex comprises two membrane proteins, a glycosylated gp91phox and a non-glycosylated p22phox. Gp91phox contains all of the redox carriers necessary to reduce molecular oxygen to superoxide using NADPH. The capacity of gp91phox to produce superoxide in the absence of its membrane partner p22phox has been little studied. In the present study, we have generated in Pichia pastoris for the first time an active form of bovine gp91phox able to carry out the entire NADPH oxidase activity in the absence of p22phox. Collected information on the maturation and the activity of the recombinant gp91phox and the participation of individual cytosolic subunits in the active complex allowed us to propose, in the absence of p22phox, an unconventional stabilized complex compared with the heterodimer.