Targeted gene modification mediated by single-stranded oligonucleotides (SSOs) holds great potential for widespread use in a number of biological and biomedical fields, including functional genomics and gene therapy. By using this approach, specific genetic changes have been created in a number of prokaryotic and eukaryotic systems. In mammalian cells, the precise mechanism of SSO-mediated chromosome alteration remains to be established, and there have been problems in obtaining reproducible targeting efficiencies. It has previously been suggested that the chromatin structure, which changes throughout the cell cycle, may be a key factor underlying these variations in efficiency. This hypothesis prompted us to systematically investigate SSO-mediated gene repair at various phases of the cell cycle in a mammalian cell line. We found that the efficiency of SSO-mediated gene repair was elevated by approximately 10-fold in thymidine-treated S-phase cells. The increase in repair frequency correlated positively with the duration of SSO/thymidine coincubation with host cells after transfection. We supply evidence suggesting that these increased repair frequencies arise from a thymidine-induced slowdown of replication fork progression. Our studies provide fresh insight into the mechanism of SSO-mediated gene repair in mammalian cells and demonstrate how its efficiency may be reliably and substantially increased.