The E2 ubiquitin conjugating enzyme Ubc13 and the E3 ubiquitin ligases Rad18 and Rnf8 promote homologous recombination (HR)-mediated double-strand break (DSB) repair by enhancing polymerization of the Rad51 recombinase at γ-ray-induced DSB sites. To analyze functional interactions between the three enzymes, we created RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)clones in chicken DT40 cells. To assess the capability of HR, we measured the cellular sensitivity to camptothecin (topoisomerase I poison) and olaparib (poly(ADP ribose)polymerase inhibitor) because these chemotherapeutic agents induce DSBs during DNA replication, which are repaired exclusively by HR. RAD18(-/-), RNF8(-/-) and RAD18(-/-)/RNF8(-/-) clones showed very similar levels of hypersensitivity, indicating that Rad18 and Rnf8 operate in the same pathway in the promotion of HR. Although these three mutants show less prominent defects in the formation of Rad51 foci than UBC13(-/-)cells, they are more sensitive to camptothecin and olaparib than UBC13(-/-)cells. Thus, Rad18 and Rnf8 promote HR-dependent repair in a manner distinct from Ubc13. Remarkably, deletion of Ku70, a protein essential for nonhomologous end joining (NHEJ) significantly restored tolerance of RAD18(-/-) and RNF8(-/-) cells to camptothecin and olaparib without affecting Rad51 focus formation. Thus, in cellular tolerance to the chemotherapeutic agents, the two enzymes collaboratively promote DSB repair by HR by suppressing the toxic effect of NHEJ on HR rather than enhancing Rad51 focus formation. In contrast, following exposure to γ-rays, RAD18(-/-), RNF8(-/-), RAD18(-/-)/RNF8(-/-) and UBC13(-/-)cells showed close correlation between cellular survival and Rad51 focus formation at DSB sites. In summary, the current study reveals that Rad18 and Rnf8 facilitate HR by two distinct mechanisms: suppression of the toxic effect of NHEJ on HR during DNA replication and the promotion of Rad51 focus formation at radiotherapy-induced DSB sites.