Activation of interferon regulatory factor (IRF)-3 and/or IRF-7 drives the expression of antiviral genes and the production of alpha/beta IFN, a hallmark of antiviral responses triggered by Toll-like receptors (TLR). Here we describe a novel antiviral signaling pathway operating in myeloid (m) dendritic cells (DC) and macrophages that does not require IRF-3 and/or IRF-7 but is driven by IRF-1. IRF-1 together with myeloid differentiation factor 88 (MyD88) or IL-1 receptor-associated kinase (IRAK)-1 triggered IFN-beta promoter activation. IRF-1 physically interacted with MyD88 and activation of mDC via TLR-9 induced IRF-1-dependent IFN-beta production paralleled by rapid transcriptional activation of IFN-stimulated genes. The NF-kappaB-dependent production of pro-inflammatory cytokines, however, was not influenced by IRF-1. TLR-9 signaling through this pathway conferred cellular antiviral resistance while IRF-1-deficient mice displayed enhanced susceptibility to viral infection. These results demonstrate that TLR-9 activation of mDC and macrophages contributes to antiviral immunity via IRF-1.