Interferon (IFN)-gamma induces the expression of the gp91(phox) gene both during myeloid differentiation and also in mature phagocytes through several cis-elements and their binding proteins. To find new cis-elements for this induction, transient expression assays were performed using a reporter gene driven by serially truncated gp91(phox) promoters in U937 cells. The results suggest that a critical cis-element for induction exists in the region from bp -115 to -96 of the promoter. Site-directed mutagenesis showed that a gamma-activated sequence (GAS) element at bp -100 (-100GAS) of the gp91(phox) promoter plays a pivotal role for the IFN-gamma-dependent activity of the bp -115 to +12 region of the gp91(phox) promoter. Electrophoretic mobility shift assays using several GAS competitors and specific antibodies indicated that phosphorylated STAT-1alpha specifically binds to the -100GAS. Site-directed mutagenesis showed that an interferon-stimulated response element (ISRE) at bp -88 (-88ISRE) mediates the induction of the gene by IFN-gamma in cooperation with -100GAS. Electrophoretic mobility shift assay showed that IRF-1 dominantly binds to -88ISRE in an IFN-gamma-dependent fashion. These results demonstrate a new mechanism for IFN-gamma-induced transcription of the gp91(phox) gene by the cooperation of STAT-1alpha and IRF-1 binding to -100GAS and -88ISRE, respectively.