The p53 tumour-suppressor protein is a sequence-specific DNA-binding transcription factor that induces cell cycle arrest or apoptosis in response to genotoxic stress. Activation of p53 by DNA-damaging agents is critical for eliminating cells with damaged genomic DNA and underlies the apoptotic response of human cancers treated with ionizing radiation (IR) and radiomimetic drugs. The molecular mechanisms by which DNA damage activates p53 have not been elucidated. Both the levels of p53 protein and its affinity for specific DNA sequences increase in response to genotoxic stress. In vitro, the affinity of p53 for DNA is regulated by its carboxy-terminus. We therefore examined whether this region of p53 is targeted by DNA-damage signalling pathways in vivo. In nonirradiated cells, serines 376 and 378 of p53 were phosphorylated. IR led to dephosphorylation of Ser376, creating a consensus binding site for 14-3-3 proteins and leading to association of p53 with 14-3-3. In turn, this increased the affinity of p53 for sequence-specific DNA. Consistent with the lack of p53 activation by IR in ataxia telangiectasia (AT; refs 14,15), neither Ser376 dephosphorylation, nor the interaction of p53 with 14-3-3 proteins occurred in AT cells.