The human inducible NOS (iNOS) promoter transcriptionally regulated by 5' flanking region extending 16 kb upstream that contains cytokine-responsive DNA motifs. In this study, we further identified a classic inducible enhancer located between -5 and -6 kb in the hiNOS upstream promoter. This 1 kb promoter sequence functions as a cytokine-inducible enhancer in an orientation- and position-independent manner in human lung A549 and liver AKN1 cells. This DNA enhancer also confers cytokine inducibility to the heterologous thymidine kinase (TK) promoter. Chromatin immunoprecipitation (ChIP) analysis was applied, and confirmed cytokine-inducible in vivo DNA-protein interactions within this enhancer region. In vivo functional binding of both NF-kappaB (p65/p50) and Stat-1alpha at the -5.8 kb human iNOS promoter site was significantly increased in A549 cells after cytokine stimulation, while only Stat-1alpha bound at the -5.2 kb site. These results identify the -5 to -6 kb promoter region as a classic transcriptional enhancer for the human iNOS gene and provide definitive in vivo evidence of specific NF-kappaB and Stat-1 nuclear protein binding that mediates transcription of the hiNOS gene under cytokine stimulation.