The activity of the inducible nitric oxide synthase enzyme (iNOS) is tightly controlled, partly at the transcriptional level. We find NF-kappa B/Rel activation (p50-p50 and p50-p65) in RAW 264.7 macrophages after lipopolysaccharide treatment and binding to both NF-kappa B sites in the mouse iNOS promoter. To delineate the importance of NF-kappa B/Rel in iNOS gene transcription, we used an unusually direct approach to try to improve on the antioxidant-treatment or reporter techniques, namely the depletion of NF-kappa B/Rel activity through the use of a phosphorothioate-modified oligonucleotide containing three copies of the NF-kappa B consensus sequence. The reduction in NF-kappa B/Rel activity (particularly that binding to the downstream of the two sites) was associated with a 50% reduction in NO output and a reduction in the quantity of the iNOS protein expressed. These results point to the probability that physiologically relevant NF-kappa B/Rel activators or repressors other than lipopolysaccharide might crucially affect the macrophage NO response.