Interferon-γ (IFN-γ) is required for induction of the human nitric oxide synthase-2 (NOS2) gene in lung epithelium. Although the human NOS2 promoter region contains many cytokine-responsive elements, the molecular basis of induction is only partially understood. Here, the major cis-regulatory elements that control IFN-γ-inducible NOS2 gene transcription in human lung epithelial cells are identified as composite response elements that bind signal transducer and activator of transcription 1 (STAT-1) and activator protein 1 (AP-1), which is comprised of c-Fos, Fra-2, c-Jun, and JunD. Notably, IFN-γ activation of the human NOS2 promoter is shown to require functional AP-1 regulatory region(s), suggesting a role for AP-1 activation/binding in the IFN-γ induction of genes. We show that c-Fos interacts with STAT-1 after IFN-γ activation and the c-Fos/STAT-1 complex binds to the γ-activated site (GAS) element in close proximity to AP-1 sites located at 4.9 kb upstream of the transcription start site. Taken together, our findings support a model in which a physical interaction between c-Fos and STAT-1 participates in NOS2 gene transcriptional activation.