The Ku autoantigen is a well-characterized heterodimer of 70 and 86 kDa that binds to DNA ends, but its cellular function has been obscure. An electrophoretic mobility-shift assay and Ku antisera were used to show that Ku or a closely related protein was deficient in three mutant hamster cell lines from x-ray-sensitive complementation group 5, which is characterized by defects in DNA double-strand break repair and V(D)J recombination. Furthermore, Ku protein expression was restored when the cells reverted to x-ray resistance. The Ku p86 gene maps to human chromosome 2q33-35, and group 5 cells are rescued by almost precisely the same region, 2q34-36. Thus, biochemical and genetic evidence suggests that Ku is involved in pathways for DNA recombination and repair. By its association with a DNA-dependent protein kinase activated by DNA ends, Ku may also initiate a signaling pathway induced by DNA damage, perhaps for cell cycle arrest.