Transcriptional activation of the iNOS gene requires multiple interactions of cis-elements and trans-acting factors. Previous in vivo footprinting studies (Nuc. Acid. Res. 24:1682, 1996) of the murine iNOS gene demonstrated LPS-inducible protection of guanines in the region -904/-883, which includes an E-box motif. In this report, by using site-directed mutagenesis of the -893/-888 E-box and correlating functional assays of the mutated iNOS promoter with USF DNA-binding activities, we demonstrate the -893/-888 E-box motif is functionally required for iNOS regulation in murine mesangial cells and that upstream stimulatory factors (USFs) are in vivo components of the iNOS transcriptional response complex. Mutation of the E-box sequence augmented the iNOS response to IL-1ß in transiently transfected mesangial cells. Gel mobility shift assays demonstrated that USFs can no longer binding to the -893/-888 E-box promoter region when the E-box is mutated. Cotransfection of USF1 and USF2 expression vectors with iNOS promoter-luciferase reporter constructs suppressed IL-1ß simulated iNOS promoter activity. Cotransfection of dominant-negative USF2 mutants lacking the DNA binding domain or cis-element decoys containing concatamers of the -904/-883 region augmented IL-1ß stimulation of iNOS promoter activity. Using gel mobility shift assays, only USF1 and USF2 supershifted the USF protein/DNA complexes. These results demonstrated that USF binding to the E-box at -893/-888 serves to trans-repress basal and IL-1ß induction of the iNOS promoter.