NADPH oxidase 1 (Nox1), a homolog of gp91(phox), is dominantly expressed in large intestinal epithelium, and reactive oxygen species derived from Nox1 are suggested to serve a role in host defense. We report that interferon (IFN)-gamma, a crucial transactivator of the gp91(phox) gene, also stimulates expression of Nox1 mRNA and protein in large intestinal epithelium (T84 cells), leading to fourfold upregulation of superoxide anion (O(2)(-)) generation. Introduction of small interfering Nox1 RNA completely blocked this priming. We cloned the region from -4,831 to +195 bp of the human Nox1 gene. To reveal IFN-gamma-responsive cis elements, we performed transient expression assays using a reporter gene driven by serially truncated Nox1 promoters in T84 cells. IFN-gamma-responsive elements were located between -4.3 and -2.6 kb, and one gamma-activated sequence (GAS) element present at -3,818 to -3,810 bp exhibited this IFN-gamma-dependent promoter activity. IFN-gamma caused tyrosine phosphorylation of signal transducer and activator of transcription 1 (STAT1) and produced a protein-GAS complex that was recognized by anti-STAT1 antibody. The introduction of three-point mutation of GAS, which did not interact with STAT1, completely canceled the IFN-gamma-dependent promoter activity of the region from -4,831 to +195 bp. A Janus protein tyrosine kinase 2 inhibitor (AG490) blocked the IFN-gamma-stimulated tyrosine phosphorylation of STAT1, promoter activity of the -4,831 to +195 bp region, Nox1 mRNA expression, and O(2)(-) production, also suggesting a crucial role of STAT1 and GAS in the IFN-gamma-stimulated transcription of the Nox1 gene. Our results support a potential contribution of Nox1 to mucosal host defense and inflammation in the colon.