We have isolated a genomic clone of the human IRF-3 gene containing 779 nucleotides of the 5' flanking region and the complete intron exon sequence. The gene has eight exons which span about 6 kb on chromosome 19q13.3. The IRF-3 promoter has neither a conserved TATA box nor a CCAAT box motif but is GC rich. Several putative DNA-binding elements were identified, including three SP-1 sites, a USF element, a HOX box, a CarG box, and an NF-1 site. Deletion analysis of the promoter region showed that the core basal promoter, consisting of 113 bp 5' of the first transcription start site, was sufficient for constitutive expression. This region contains only one of the SP-1 sites as well as the HOX element and NF-1 site, and although it is GC rich, it does not contain any of the other putative DNA-binding sites. In contrast, the mouse IRF-3 promoter, while displaying a high degree of homology with the human promoter, contains both TATA and CCAAT box motifs, suggesting that, at least at the level of transcription initiation, these genes may be differentially regulated.