Oligonucleotides (ODNs) can direct the exchange of single nucleotides at specific sites in the mammalian genome. It is generally believed that the ODN aligns in homologous register with its complementary site in the target gene and provides a template for the endogenous repair machinery to alter the sequence of the gene. We have been studying the initial phase of the reaction with particular emphasis on the cellular events that occur when the oligonucleotide enters the cell. Our results show that, following introduction of the oligonucleotide, the DNA-damage response pathway is activated, evidenced by the presence of phosphorylated p53, Chk1 and Chk2, respectively. As a result, progression of some of these cells through the cell cycle is slowed and those bearing corrected genes all contain phosphorylated Chk1 and Chk2. In contrast, uncorrected cells contain much lower levels of these proteins in the activated state and pass through the cell cycle in a normal fashion. We suggest that gene repair directed by oligonucleotides activates a pathway that prevents corrected cells from proliferating in cell culture through the activation of Chk1 and Chk2. Our results impact the future use of gene repair for ex vivo gene therapy applications.