DNA double-strand breaks (DSBs) are the most biologically damaging lesions caused by ionizing radiation (IR) and some chemicals. They occur during DNA replication or as initiators of programmed processes like V(D)J recombination and meiotic exchange. If not repaired, DSBs can lead to cell cycle arrest, apoptosis, or mitotic death, or cause genomic rearrangements leading to cancer. Higher eukaryotic cells primarily repair DSBs through nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ repairs DSBs without sequence homology, while HR uses homology for accurate repair. 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. DSBs from replication inhibitors like aphidicolin are repaired only by HR. This study shows that NHEJ is important in all cell cycle phases, while HR is most critical in late S/G₂. HR is also involved in repairing replication-associated DSBs. The study used γ-H2AX foci to monitor DSB repair and found that NHEJ is the main repair pathway in G₁, while HR is more important in late S/G₂. HR is essential for repairing replication-associated DSBs, while NHEJ is more important for IR-induced DSBs in the S phase. The study also shows that HR is important for radioresistance in late S/G₂, while NHEJ is important for survival in all phases. The findings suggest that HR and NHEJ have different roles in DSB repair depending on the cell cycle phase and the type of DSB. The study provides the first cell cycle-specific evaluation of the contributions of NHEJ and HR to DSB repair after a radiation dose of 1 Gy.DNA double-strand breaks (DSBs) are the most biologically damaging lesions caused by ionizing radiation (IR) and some chemicals. They occur during DNA replication or as initiators of programmed processes like V(D)J recombination and meiotic exchange. If not repaired, DSBs can lead to cell cycle arrest, apoptosis, or mitotic death, or cause genomic rearrangements leading to cancer. Higher eukaryotic cells primarily repair DSBs through nonhomologous end joining (NHEJ) or homologous recombination (HR). NHEJ repairs DSBs without sequence homology, while HR uses homology for accurate repair. 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. DSBs from replication inhibitors like aphidicolin are repaired only by HR. This study shows that NHEJ is important in all cell cycle phases, while HR is most critical in late S/G₂. HR is also involved in repairing replication-associated DSBs. The study used γ-H2AX foci to monitor DSB repair and found that NHEJ is the main repair pathway in G₁, while HR is more important in late S/G₂. HR is essential for repairing replication-associated DSBs, while NHEJ is more important for IR-induced DSBs in the S phase. The study also shows that HR is important for radioresistance in late S/G₂, while NHEJ is important for survival in all phases. The findings suggest that HR and NHEJ have different roles in DSB repair depending on the cell cycle phase and the type of DSB. The study provides the first cell cycle-specific evaluation of the contributions of NHEJ and HR to DSB repair after a radiation dose of 1 Gy.