53BP1 participates in the cellular response to DNA damage. Like many proteins involved in the DNA damage response, 53BP1 becomes hyperphosphorylated after radiation and colocalizes with phosphorylated H2AX in megabase regions surrounding the sites of DNA strand breaks. However, it is not yet clear whether the phosphorylation status of 53BP1 determines its localization or vice versa. In this study we mapped a region upstream of the 53BP1 C terminus that is required and sufficient for the recruitment of 53BP1 to these DNA break areas. In vitro assays revealed that this region binds to phosphorylated but not unphosphorylated H2AX. Moreover, using H2AX-deficient cells reconstituted with wild-type or a phosphorylation-deficient mutant of H2AX, we have shown that phosphorylation of H2AX at serine 140 is critical for efficient 53BP1 foci formation, implying that a direct interaction between 53BP1 and phosphorylated H2AX is required for the accumulation of 53BP1 at DNA break sites. On the other hand, radiation-induced phosphorylation of the 53BP1 N terminus by the ATM (ataxia-telangiectasia mutated) kinase is not essential for 53BP1 foci formation and takes place independently of 53BP1 redistribution. Thus, these two damage-induced events, hyperphosphorylation and relocalization of 53BP1, occur independently in the cell.The DNA of eukaryotic cells is constantly exposed to endogenous and exogenous DNA-damaging agents. To prevent the accumulation of genomic damage and avert cellular dysfunction, cells have evolved complex response mechanisms. 53BP1 was initially identified as a protein that binds to the central DNA binding domain of p53 and enhances p53-mediated transcriptional activation (1, 2). In response to genotoxic stress, 53BP1 rapidly redistributes from a diffuse nuclear localization into distinct nuclear foci suggesting that 53BP1 is involved in the DNA damage response (3-6). Moreover the C terminus of 53BP1 contains two BRCT domains, a motif found in a number of proteins implicated in various aspects of cell cycle control, recombination, and DNA repair (7,8). Subsequent studies have shown that 53BP1 becomes hyperphosphorylated in response to ionizing radiation (IR) 1 and colocalizes with phosphorylated histone H2AX (␥-H2AX) at the sites of DNA lesions (3, 4). Other proteins known to be involved in the DNA damage signaling pathway (i.e. BRCA1, Rad51, NBS1, and TopoBP1) were also found to colocalize with 53BP1 in these inducible foci (3,4,6,9). Direct evidence for an important role of 53BP1 in the DNA damage response came recently from studies using 53BP1-deficient cells. Human cell lines treated with specific small interfering RNA to silence 53BP1 expression exhibited a defect in the intra-S phase checkpoint and, at low IR doses, a partial defect in the G 2 -M checkpoint (10 -12). Moreover 53BP1-deficient mice are hypersensitive to ionizing radiation and show an increased incidence of developing thymic lymphomas (13). Several lines of evidence suggest that 53BP1 is a substrate of ATM, the kinase mutated in the hu...