Potentially lethal damage (PLD) repair has been defined as that property conferring the ability of cells to recover from DNA damage depending on the postirradiation environment. Using a novel cyclin dependent kinase 1 inhibitor RO-3306 to arrest cells in the G₂ phase of the cell cycle, examined PLD repair in G₂ in cultured Chinese hamster ovary (CHO) cells. Several CHO-derived DNA repair mutant cell lines were used in this study to elucidate the mechanism of DNA double-strand break repair and to examine PLD repair during the G₂ phase of the cell cycle. While arrested in G₂ phase, wild-type CHO cells displayed significant PLD repair and improved cell survival compared with cells released immediately from G₂ after irradiation. Both the radiation-induced chromosomal aberrations and the delayed entry into mitosis were also reduced by G₂-holding PLD recovery. The PLD repair observed in G₂ was observed in nonhomologous end-joining (NHEJ) mutant cell lines but absent in homologous recombination mutant cell lines. From the survival curves, G₂-NHEJ mutant cell lines were found to be very sensitive to gamma-ray exposure when compared to G₂/homologous recombination mutant cell lines. Our findings suggest that after exposure to ionizing radiation during G₂, NHEJ is responsible for the majority of non-PLD repair, and conversely, that the homologous recombination is responsible for PLD repair in G₂.