The phagocyte respiratory burst oxidase is a flavin-adenine dinucleotide (FAD)-dependent dehydrogenase and an electron transferase that reduces molecular oxygen to superoxide anion, a precursor of microbicidal oxidants. Several proteins required for assembly of the oxidase have been characterized, but the identity of its flavin-binding component has been unclear. Oxidase activity was reconstituted in vitro with only the purified oxidase proteins p47^phox, p67^phox, Rac-related guanine nucleotide (GTP)-binding proteins, and membrane-bound cytochrome b₅₅₈. The reconstituted oxidase required added FAD, and FAD binding was localized to cytochrome b₅₅₈. Alignment of the amino acid sequence of the β subunit of cytochrome b₅₅₈ (gp91^phox) with other flavoproteins revealed similarities to the nicotinamide adenine dinucleotide phosphate (reduced) (NADPH)-binding domains. Thus flavocytochrome b₅₅₈ is the only obligate electron transporting component of the NADPH oxidase.