We have examined the consequences of overexpression of the IκBα and IκBβ inhibitory proteins on the regulation of NF-κB-dependent beta interferon (IFN-β) gene transcription in human cells after Sendai virus infection. In transient coexpression studies or in cell lines engineered to express different forms of IκB under tetracycline-inducible control, the IFN-β promoter (−281 to +19) linked to the chloramphenicol acetyltransferase reporter gene was differentially inhibited in response to virus infection. IκBα exhibited a strong inhibitory effect on virus-induced IFN-β expression, whereas IκBβ exerted an inhibitory effect only at a high concentration. Despite activation of the IκB kinase complex by Sendai virus infection, overexpression of the double-point-mutated (S32A/S36A) dominant repressors of IκBα (TD-IκBα) completely blocked IFN-β gene activation by Sendai virus. Endogenous IFN-β RNA production was also inhibited in Tet-inducible TD-IκBα-expressing cells. Inhibition of IFN-β expression directly correlated with a reduction in the binding of NF-κB (p50-RelA) complex to PRDII after Sendai virus infection in IκBα-expressing cells, whereas IFN-β expression and NF-κB binding were only slightly reduced in IκBβ-expressing cells. These experiments demonstrate a major role for IκBα in the regulation of NF-κB-induced IFN-β gene activation and a minor role for IκBβ in the activation process.