Nitric oxide synthase (NOS) activity was induced in the cytosol of A-172 cells by treatment with lipopolysaccharide, tumor necrosis factor-alpha, and interferon-gamma. A 130-kDa human inducible NOS (iNOS) protein was detected with anti-rat iNOS antibody by western blot analysis. Northern blot analysis showed that the iNOS mRNA was approximately 4.5 kb, using a cDNA fragment for human iNOS, isolated from stimulated A-172 cells by reverse transcriptase-PCR, as a probe. The mRNA was induced by interferon-gamma at a trace level, and its expression was synergistically enhanced by simultaneous addition of lipopolysaccharide, tumor necrosis factor-alpha, and, to a larger extent, interleukin-1 beta. The mRNA expression was blocked by coincubation with actinomycin D or cycloheximide. Furthermore, by transfecting the mouse iNOS gene promoter into A-172 cells, transcriptional activation of the iNOS gene was detected in these cells upon stimulation with lipopolysaccharide and cytokines. The pattern of promoter activation correlated well with that of iNOS mRNA expression upon stimulation. These data indicate that expression of iNOS is transcriptionally regulated in A-172 cells. This process requires de novo protein synthesis with a mechanism similar to that in place in mouse macrophages.