The inducible isoform of nitric oxide (NO) synthase (iNOS) has been implicated in the development of inflammatory tissue injury. Stimulation of the transformed intestinal epithelial cell line DLD-1 by interleukin-1β and interferon-γ resulted in an increase in NO production and iNOS expression. Northern hybridization analysis of cytokine-stimulated cells revealed a 20-fold increase in the steady-state level of iNOS mRNA. Nuclear run-on analysis demonstrated a 2-4 fold increase in the transcription of iNOS mRNA. Cycloheximide fully inhibited the cytokine-mediated increase in iNOS transcription, suggesting that de novo protein synthesis was required for transcriptional activation. A genomic clone containing exons 1 and 2 of the human gene and 13.1 kb of the 5′ flanking region was isolated and used to develop a series of promoter deletion constructs linked to a luciferase reporter vector. Cytokine-responsive elements were identified between -8.7 to -10.7 kb 5′ to the transcription inititation site. Insertion of this 2 kb region into a reporter construct terminating at -1.1 kb, in the reverse and forward orientation, demonstrated cytokine responsiveness equivalent to that seen with the -10.7 kb construct. Thus, our data indicate for the first time that human iNOS expression is regulated by a cytokine-responsive enhancer. DNA sequencing of this 2 kb region revealed the presence of several consensus sites for cytokine-responsive transcription factors.