Viral infection and stimulation with lipopolysaccharide (LPS) or double stranded RNA (dsRNA) induce phosphorylation of interferon (IFN) regulatory factor (IRF)-3 and its translocation to the nucleus, thereby leading to the IFN-beta gene induction. Recently, two IkappaB kinase (IKK)-related kinases, inducible IkappaB kinase (IKK-i) and TANK-binding kinase 1 (TBK1), were suggested to act as IRF-3 kinases and be involved in IFN-beta production in Toll-like receptor (TLR) signaling and viral infection. In this work, we investigated the physiological roles of these kinases by gene targeting. TBK1-deficient embryonic fibroblasts (EFs) showed dramatic decrease in induction of IFN-beta and IFN-inducible genes in response to LPS or dsRNA as well as after viral infection. However, dsRNA-induced expression of these genes was residually detected in TBK1-deficient cells and intact in IKK-i-deficient cells, but completely abolished in IKK-i/TBK1 doubly deficient cells. IRF-3 activation, in response not only to dsRNA but also to viral infection, was impaired in TBK1-deficient cells. Together, these results demonstrate that TBK1 as well as, albeit to a lesser extent, IKK-i play a crucial role in the induction of IFN-beta and IFN-inducible genes in both TLR-stimulated and virus-infected EFs.