Nuclear factor-kappaB (NF-kappaB) and p53 critically determine cancer development and progression. Defining the cross talk between these transcription factors can expand our knowledge on molecular mechanisms of tumorigenesis. Here, we show that induction of replicational stress activates NF-kappaB p65 and triggers its interaction with p53 in the nucleus. Experiments with knockout cells show that p65 and p53 are both required for enhanced NF-kappaB activity during S-phase checkpoint activation involving ataxia-telangiectasia mutated and checkpoint kinase-1. Accordingly, the pro-inflammatory cytokine tumor necrosis factor-alpha (TNF-alpha) also triggers formation of a transcriptionally active complex containing nuclear p65 and p53 on kappaB response elements. Gene expression analyses revealed that, independent of NF-kappaB activation in the cytosol, TNF-induced NF-kappaB-directed gene expression relies on p53. Hence, p53 is unexpectedly necessary for NF-kappaB-mediated gene expression induced by atypical and classical stimuli. Remarkably, data from gain- and loss-of function approaches argue that anti-apoptotic NF-kappaB p65 activity is constitutively evoked by a p53 hot-spot mutant frequently found in tumors. Our observations suggest explanations for the outstanding question why p53 mutations rather than p53 deletions arise in tumors of various origins.