[en] IKK alpha regulates many chromatin events in the nuclear phase of the NF-kappa B program, including phosphorylation of histone H3 and removal of co-repressors from NF-kappa B-dependent promoters. However, all of the nuclear functions of IKK alpha are not understood. In this study, using mouse embryonic fibroblasts IKK alpha knock-out and reexpressing IKK alpha after retroviral transduction, we demonstrate that IKK alpha contributes to NF-kappa B/p65 DNA binding activity on an exogenous kappa B element and on some, but not all, endogenous NF-kappa B-target promoters. Indeed, p65 chromatin immunoprecipitation assays revealed that IKK alpha is crucial for p65 binding on kappa B sites of icam-1 and mcp-1 promoters but not on i kappa b alpha promoter. The mutation of IKK alpha putative nuclear localization sequence, which prevents its nuclear translocation, or of crucial serines in the IKK alpha activation loop completely inhibits p65 binding on icam-1 and mcp-1 promoters and rather enhances p65 binding on the i kappa b alpha promoter. Further molecular studies demonstrated that the removal of chromatin-bound HDAC3, a histone deacetylase inhibiting p65 DNA binding, is differentially regulated by IKK alpha in a promoter-specific manner. Indeed, whereas the absence of IKK alpha induces HDAC3 recruitment and repression on the icam-1 promoter, it has an opposite effect on the i kappa b alpha promoter, where a better p65 binding occurs. We conclude that nuclear IKK alpha is required for p65 DNA binding in a gene-specific manner.