The phagocyte NADPH-dependent oxidase generates superoxide (O 2.-) by reducing molecular oxygen through flavocytochrome b558 (flavocytochrome b), a heterodimeric oxidoreductase composed of gp91phox and p22phox subunits. Although each flavocytochrome b molecule contains two heme groups, their precise distribution within the heterodimer is unknown. Among functionally and/or structurally related oxidoreductases, histidines at codons 101, 111, 115, 119, 209, 210, and 222 of gp91phox are conserved and potential candidates to ligate heme. We compared biochemical and functional features of normal flavocytochrome b with those in cells expressing gp91phox harboring amino acid substitutions at each of these histidines. Surface expression of flavocytochrome b and heterodimer formation were relatively unaffected in cells expressing gp91phox H111L, H119L, or H210L. These mutations also had no effect on the flavocytochrome b heme spectrum, although NADPH oxidase activity was decreased in cells expressing gp91phox H119L or H210L. In contrast, gp65 was not processed to gp91phox, heterodimers did not form, and flavocytochrome b was not expressed on the surface of cells expressing gp91phox H101L, H115L, H115D, H209C, H209Y, H222L, H222C, or H222R. Similarly, this subset of mutants lacked detectable O2 .--generating activity, and flavocytochrome b purified from these cells contained little or no heme. These findings demonstrate that His 101, His115, His209, and His222 of gp91phox are critical for heme binding and biosynthetic maturation of flavocytochrome b.