Nuclear factor κB (NF-κB)/Rel transcription factors are key regulators of a variety of genes involved in immune and inflammatory responses, growth, differentiation, apoptosis, and development. In unstimulated cells, NF-κB/Rel proteins are sequestered in the cytoplasm by IκB inhibitor proteins. Many extracellular stimuli, such as tumor necrosis factor α (TNFα), cause rapid phosphorylation of IκB at N-terminal serine residues leading to ubiquitination and degradation of the inhibitor. Subsequently, NF-κB proteins translocate to the nucleus and activate gene expression through κB response elements. TNFα, as well as certain other stimuli, also induces the phosphorylation of the NF-κB proteins. Previously, we have shown that TNFα induces RelA/p65 phosphorylation at serine 529 and that this inducible phosphorylation increases NF-κB transcriptional activity on an exogenously supplied reporter (1). In this report, we demonstrate that casein kinase II (CKII) interacts with p65 in vivo and can phosphorylate p65 at serine 529 in vitro. A CKII inhibitor (PD144795) inhibited TNFα-induced p65 phosphorylation in vivo. Furthermore, our results indicate that the association between IκBα and p65 inhibits p65 phosphorylation by CKII and that degradation of IκBα allows CKII to phosphorylate p65 to increase NF-κB transactivation potential. These data may explain the ability of CKII to modulate cell growth and demonstrate a mechanism whereby CKII can function in an inducible manner.