Transcription of the murine interferon-A4 (IFN-A4) gene is mediated by a virus responsive element (VRE-A4) located in the promoter proximal [-120 to -43] region. VRE-A4 contains four DNA modules (A to D) which cooperate for maximal IFN-A4 activation following virus infection. The differential expression between the highly expressed IFN-A4 and the weakly inducible IFN-A11 gene promoters is essentially due to point mutations within the C and D modules of the virus-responsive element VRE-A11. We now demonstrate that in murine L929 and human 293 cells, transcription factors IRF-3 and IRF-7, which are potent activators of virus-induced type I IFN transcription, differentially affect IFN-A4 and IFN-A11 promoter activities. Using electrophoretic mobility shift assays and DNase I footprinting data, our studies demonstrate that the AB modules correspond to a preferential site for IRF-7, whereas the C module is preferentially recognized by IRF-3. Furthermore, transfection of reporter constructs driven by four copies of different GAAANN hexameric motifs found within VRE-A4 indicates that the NN residues of these hexameric sequences define the preferential binding sites for IRF-3 or IRF-7. Together, these experiments clarify the molecular basis for differential expression of IFN-A genes following virus infection by delineating the sequence requirements for IRF association with the virus responsive elements of the IFN-A genes.