The p53 tumor suppressor protein preserves genome integrity by regulating growth arrest and apoptosis in response to DNA damage. In response to ionizing radiation (IR), ATM, the gene product mutated in ataxia telangiectasia, stabilizes and activates p53 through phosphorylation of Ser15and (indirectly) Ser20. Here we show that phosphorylation of p53 on Ser46, a residue important for p53 apoptotic activity, as well as on Ser9, in response to IR also is dependent on the ATM protein kinase. IR-induced phosphorylation at Ser46was inhibited by wortmannin, a phosphatidylinositol 3-kinase inhibitor, but not PD169316, a p38 MAPK inhibitor. p53 C-terminal acetylation at Lys320and Lys382, which may stabilize p53 and activate sequence-specific DNA binding, required Ser15phosphorylation by ATM and was enhanced by phosphorylation at nearby residues including Ser6, Ser9, and Thr18. These observations, together with the proposed role of Ser46phosphorylation in mediating apoptosis, suggest that ATM is involved in the initiation of p53-dependent apoptosis after IR in human lymphoblastoid cells.