Inducible nitric oxide synthase (iNOS) is involved in many physiological and pathophysiological processes, including septic shock and acute kidney failure. Little is known about transcriptional regulation of the human iNOS gene in vivo under basal conditions or in sepsis. Accordingly, we developed transgenic mice carrying an insertional human iNOS promoter-reporter gene construct. In these mice, the proximal 8.3 kb of the human iNOS 5'-flanking region controls expression of the reporter gene of enhanced green fluorescent protein (EGFP). Patterns of human iNOS promoter/EGFP transgene expression in tissues were examined by fluorescence microscopy and immunoblotting. Endogenous murine iNOS was basally undetectable in kidney, intestine, spleen, heart, lung, liver, stomach, or brain. In contrast, EGFP from the transgene was basally expressed in kidney, brain, and spleen, but not the other tissues of the transgenic mice. Bacterial lipopolysaccharide induced endogenous iNOS expression in kidney, intestine, spleen, lung, liver, stomach, and heart, but not brain. In contrast, human iNOS promoter/EGFP transgene expression was induced above basal levels only in intestine, spleen, brain, stomach, and lung. Within kidney, human iNOS promoter/EGFP fluorescence was detected most prominently in proximal tubules of the outer cortex and collecting ducts and colocalized with endogenous mouse iNOS. Within the collecting duct, both endogenous iNOS and the human iNOS promoter/EGFP transgene were expressed in cells lacking aquaporin-2 immunoreactivity, consistent with expression in intercalated cells. Although it remains possible that essential regulatory elements reside in remote locations of the gene, our data concerning this 8.3-kb region provide the first in vivo evidence suggesting differential transcriptional control of the human iNOS gene in these organs and marked differences in transcriptional regulatory regions between the murine and human genes.