The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between -5.2 and -6.1 kb in the 5'-flanking hiNOS promoter DNA, which contains multiple nuclear factor kappa beta (NF-kappa B) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-alpha, interleukin-1 beta, and IFN-gamma (TNF-alpha, IL-1 beta, and IFN-gamma) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at -5.8 kb in the hiNOS promoter identifies a bifunctional NF-kappa B/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at -5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-kappa B activates hiNOS promoter-reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-kappa B function in these cells. These results indicate that both Stat 1 and NF-kappa B are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.