Phosphorylation of nuclear factor-kappa B (NF-kappa B) subunits emerges as a mechanism by which transcriptional activity of nuclear NF-kappa B complexes is regulated in an inhibitor kappa B-independent fashion. As the main transactivator, the p65 subunit of NF-kappa B has an outstanding position in the hierarchy of NF-kappa B proteins. p65 is a multiply phosphorylated protein with phosphorylation sites in the C-terminal transactivation domain and the N-terminal Rel homology domain (RHD). In this study, we describe two previously non-reported phospho-acceptor sites within the p65 RHD. We show that differential phosphorylation of serine residues within the RHD modulates transcriptional activity in a cis-acting element and promoter-specific context, thus leading to a phosphorylation state-dependent gene expression profile. RelA(-/-) mouse embryonic fibroblasts reconstituted with wild-type p65 or p65 phosphorylation-deficient mutants showed a distinctive expression profile of synthetic kappa B-dependent reporters as well as endogenous genes. Hypophosphorylated p65 did not display cis-acting element-specific changes in DNA binding or dimerization behavior. This study shows for the first time that site-specific phosphorylation can target a transcription factor to a particular subset of genes.