Phosphorylation of the p65 subunit of NF-kappaB is required for its transcriptional activity. Recent reports show that phosphorylation of p65 at serine 276 regulates only a subset of genes, such as those encoding IL-6, IL-8, Gro-beta, and ICAM-1. In order to identify additional genes regulated by serine 276 phosphorylation, HepG2 hepatoma cells were infected with adenoviruses encoding either wild-type p65 or the S276A mutant of p65, followed by DNA microarray analysis. The results show that mutation of serine 276 affected the expression of several genes that encode proteins involved in cell cycle regulation, signal transduction, transcription, and metabolism. Notably, expression of S276A increased the mRNA and protein level of p27, a cell cycle inhibitory protein, which led to an increased association of p27 with cdk2, and inhibition of cdk2 activity. Furthermore, while wild-type NF-kappaB is known to increase cell proliferation in a number of different cancer cell lines, our data shows that S276A inhibited cell proliferation. Evidence is mounting that NF-kappaB plays a pivotal role in oncogenesis. Therapeutic agents that regulate the phosphorylation of serine 276 and p27 gene expression, therefore, may be useful as anti-cancer agents in the future.