This study investigates the contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation (IR). Using immunofluorescence detection of γ-H2AX nuclear foci, the authors show that NHEJ-defective hamster cells (CHO mutant V3) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells exhibit a minor repair defect in G1, greater impairment in S, and a substantial defect in late S/G2. The radiosensitivity of irs1SF cells is slightly higher in G1 but dramatically higher in late S/G2, while V3 cells show high sensitivity throughout the cell cycle. These findings indicate that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G2, where both pathways contribute to repair and radioresistance. Additionally, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR, and irs1SF cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.This study investigates the contributions of nonhomologous end joining (NHEJ) and homologous recombination (HR) to DNA double-strand break (DSB) repair in different cell cycle phases after physiologically relevant doses of ionizing radiation (IR). Using immunofluorescence detection of γ-H2AX nuclear foci, the authors show that NHEJ-defective hamster cells (CHO mutant V3) have strongly reduced repair in all cell cycle phases after 1 Gy of irradiation. In contrast, HR-defective CHO irs1SF cells exhibit a minor repair defect in G1, greater impairment in S, and a substantial defect in late S/G2. The radiosensitivity of irs1SF cells is slightly higher in G1 but dramatically higher in late S/G2, while V3 cells show high sensitivity throughout the cell cycle. These findings indicate that NHEJ is important in all cell cycle phases, while HR is particularly important in late S/G2, where both pathways contribute to repair and radioresistance. Additionally, DSBs produced by the replication inhibitor aphidicolin are repaired entirely by HR, and irs1SF cells show hypersensitivity to aphidicolin treatment. These data provide the first evaluation of the cell cycle-specific contributions of NHEJ and HR to the repair of radiation-induced versus replication-associated DSBs.