The phagocyte nicotinamide adenine dinucleotide phosphate (reduced form) (NADPH) oxidase was functionally reconstituted in monkey kidney COS-7 cells by transfection of essential subunits, gp91^phox, p22^phox, p47^phox, and p67^phox. COS-7 cells express the essential small guanosine 5′-triphosphatase, Rac1. Transgenic COS-phox cells were capable of arachidonic acid–induced NADPH oxidase activity up to 80% of that of human neutrophils, and of phorbol myristate acetate (PMA)–induced activity up to 20% of that of neutrophils. Expression of all 4 phox components was required for enzyme activity, and enzyme activation was associated with membrane translocation of p47^phox, p67^phox, and Rac1. Expression of p47^phox Ser303Ala/Ser304Ala or Ser379Ala phosphorylation-deficient mutants resulted in significantly impaired NAPDH oxidase activity, compared with expression of wild-type p47^phox or the p47^phox Ser303Glu/Ser304Glu phosphorylation mimic, suggesting that p47^phoxphosphorylation contributes to enzyme activity in the COS system, as is the case in neutrophils. Hence, COS-phox cells should be useful as a new whole-cell model that is both capable of high-level superoxide production and readily amenable to genetic manipulation for investigation of NADPH oxidase function. PMA-elicited superoxide production in COS-phox cells was regulated by activation of protein kinase C (PKC) and Rac. Although COS-7 cells differ from human neutrophils in PKC isoform expression, transient expression of major neutrophil isoforms in COS-phox cells did not increase PMA-induced superoxide production, suggesting that endogenous isoforms were not rate limiting. Val204 in p67^phox, previously shown to be required for NADPH oxidase activity under cell-free conditions, was found to be essential for superoxide production by intact COS-phox cells, on the basis of transfection studies using a p67^phox(Val204Ala) mutant.