The human inducible nitric oxide synthase (iNOS) gene is overexpressed in a number of human inflammatory diseases. Previously, we observed that the human iNOS gene is transcriptionally regulated by cytokines and demonstrated that the cytokine-responsive regions are upstream of -3.8 kilobase pairs (kb). Therefore, the purpose of this study was to further localize the functional enhancer elements and to assess the role of the transcription factor NF-κB in both human liver (AKN-1) and human lung (A549) epithelial cell lines. The addition of NF-κB inhibitors significantly suppressed cytokine-stimulated iNOS mRNA expression and NO synthesis, indicating that NF-κB is involved in the induction of the human iNOS gene. Analysis of the first 4.7 kb of the 5'-flanking region demonstrated basal promoter activity and failed to show any cytokine-inducible activity. However, promoter constructs extending to -5.8 and -7.2 kb revealed 2-3-fold and 4-5-fold induction, respectively, in the presence of cytokines. DNA sequence analysis from -3.8 to -7.2 kb identified five putative NF-κB cis-regulatory transcription factor binding sites upstream of -4.7 kb. Site-directed mutagenesis of these sites revealed that the NF-κB motif at -5.8 kb is required for cytokine-induced promoter activity, while the sites at -5.2, - 5.5, and -6.1 kb elicit a cooperative effect. Electromobility shift assays using a site-specific oligonucleotide and nuclear extracts from cells stimulated with cytokine-mixture, tumor necrosis factor-α or interleukin- 1β, but not interferon-γ, exhibited inducible DNA binding activity for NF- κB. These data indicate that NF-κB activation is required for cytokine induction of the human iNOS gene and identifies four NF-κB enhancer elements upstream in the human iNOS promoter that confer inducibility to tumor necrosis factor-α and interleukin-1β.