The cytochrome b heavy chain (gp91(phox)) is the redox center of the NADPH-oxidase and is highly expressed in mature myeloid cells. Point mutations at -57, -55, -53, and -52 bp of the gp91(phox) promoter have been detected in patients with chronic granulomatous disease (CGD; Newburger et al, J Clin Invest 94:1205, 1994; and Suzuki et al, Proc Natl Acad Sci USA 95:6085, 1998). We report that Elf-1 and PU. 1, ets family members highly expressed in myeloid cells, bind to this promoter element. Either factor trans-activates the -102 to +12 bp gp91(phox) promoter when overexpressed in nonhematopoietic HeLa cells or the PLB985 myeloid cell line. However, no synergy of gp91(phox) promoter activation occurs when both Elf-1 and PU.1 are overexpressed. Introduction of the -57 bp or -55 bp CGD mutations into the gp91(phox) promoter significantly reduces the binding affinity of Elf-1 and PU.1 and also reduces the ability of these factors to trans-activate the promoter. These results indicate that Elf-1 and PU.1 contribute to directing the lineage-restricted expression of the gp91(phox) gene in phagocytes and that failure of these factors to effectively interact with this promoter results in CGD.