NF-kappaB plays an important role in oncogenesis. Recently, we have demonstrated that loss of p53 function enhances DNA binding and transcriptional activities of NF-kappaB via IKKalpha and IKKbeta, and that glycolysis, activated by NF-kappaB, has an integral role in oncogene-induced cell transformation. Here, we show that ectopically expressed p53 induces acetylation and phosphorylation at Ser 536 of p65, an NF-kappaB component, and enhances DNA-binding activity of NF-kappaB. However, activated p53 suppresses transcriptional activity of NF-kappaB. Under non-stimulating conditions, p65 formed a complex with IKKalpha and IKKbeta. Activated p53 bound to p65 on DNA and disrupted binding of p65 to IKKbeta. Moreover, histone H3 kinase activity, which requires transcriptional activation of NF-kappaB, was diminished by p53. Thus, activated p53 may suppress transcriptional activity of NF-kappaB through inhibition of IKK and histone H3 kinase on DNA, suggesting a novel p53-mediated suppression system for tumorigenesis.