Abstract: ERCC1-XPF endonuclease is required for nucleotide excision repair (NER) of helix-distorting DNA lesions. However, mutations in ERCC1 or XPF in humans or mice cause a more severe phenotype than absence of NER, prompting a search for novel repair activities of the nuclease. In Saccharomyces cerevisiae, orthologs of ERCC1-XPF (Rad10-Rad1) participate in the repair of double-strand breaks (DSB). Rad10-Rad1 contributes to two error-prone DSB repair pathways: microhomology-mediated end-joining (a Ku86-independent mechanism) and single-strand annealing. To determine if ERCC1-XPF participates in DSB repair in mammals, mutant cells and mice were screened for sensitivity to gamma-irradiation. ERCC1-XPF-deficient fibroblasts were hypersensitive to gamma-irradiation and gammaH2AX foci, a marker of DSBs, persisted in irradiated mutant cells, consistent with a defect in DSB repair. Mutant mice were also hypersensitive to irradiation, establishing an essential role for ERCC1-XPF in protecting against DSBs in vivo. Mice defective in both ERCC1-XPF and Ku86 were not viable. However, Ercc1(-/-)Ku86(-/-) fibroblasts were hypersensitive to gamma-irradiation compared to single mutants and accumulated significantly greater chromosomal aberrations. Finally, in vitro repair of DSBs with 3'-overhangs led to large deletions in the absence of ERCC1-XPF. These data support the conclusion that like in yeast ERCC1-XPF facilitates DSB repair via an end-joining mechanism that is Ku86-independent.