ATM is a member of the large phosphatidylinositol-3 kinase family and plays an important role in cellular response to DNA damage. To further define the physiological roles of ATM at the cellular level, we created an isogenic set of stable cell lines differing only in their ATM status from the chicken B cell line DT40 by targeted integration. These stable DT40 cell lines, as most of transformed chicken cell lines, do not express p53. However, ATM−/− DT40 cells displayed retarded cellular proliferation, defective G 2/M checkpoint control and radio-resistant DNA synthesis. Furthermore, ATM−/− DT40 cells were sensitive to ionizing radiation and showed highly elevated frequencies of both spontaneous and radiation-induced chromosomal aberrations. In addition, a slight but significant reduction in targeted integration frequency was observed in ATM−/− DT40 cells. These results suggest that ATM has multiple p53-independent functions in cell cycle checkpoint control and in maintenance of chromosomal DNA. These ATM deficient DT40 clones therefore provide a useful model system for analysing p53-independent ATM functions.