In the 'post-genome' era, reverse genetics is one of the most informative and powerful means to investigate protein function. The chicken B lymphocyte line DT40 is widely used for reverse genetics because the cells have a number of advantages, including efficient gene targeting as well as a remarkably stable phenotype. Furthermore, the absence of functional p53 in DT40 cells enables identification of DNA damage using chromosome analysis by suppressing damage-induced apoptosis during interphase. This review summarizes the contribution of DT40 cells to reverse genetic studies of DNA damage response pathways in higher eukaryotic cells.