The p53 and NF-B transcription factor families are important, multifunctional regulators of the cellular response to stress. Here we have investigated the regulatory mechanisms controlling p53-dependent cell cycle arrest and cross talk with NF-B. Upon induction of p53 in H1299 or U-2 OS cells, we observed specific repression of cyclin D1 promoter activity, correlating with a decrease in cyclin D1 protein and mRNA levels. This repression was dependent on the proximal NF-B binding site of the cyclin D1 promoter, which has been shown to bind the p52 NF-B subunit. p53 inhibited the expression of Bcl-3 protein, a member of the IB family that functions as a transcriptional coactivator for p52 NF-B and also reduced p52/Bcl-3 complex levels. Concomitant with this, p53 induced a significant increase in the association of p52 and histone deacetylase 1 (HDAC1). Importantly, p53-mediated suppression of the cyclin D1 promoter was reversed by coexpression of Bcl-3 and inhibition of p52 or deacetylase activity. p53 therefore induces a transcriptional switch in which p52/Bcl-3 activator complexes are replaced by p52/HDAC1 repressor complexes, resulting in active repression of cyclin D1 transcription. These results reveal a unique mechanism by which p53 regulates NF-B function and cell cycle progression.