X-ray-sensitive hamster cells in complementation groups 4, 5, 6, and 7 are impaired for both double-strand break repair and V(D)J recombination. Here we show that in two mutant cell lines (XR-V15B and XR-V9B) from group 5, the genetic defects are in the gene encoding the 86-kDa subunit of the Ku autoantigen, a nuclear protein that binds to double-stranded DNA ends. These mutants express Ku86 mRNA containing deletions of 138 and 252 bp, respectively, and the encoded proteins contain internal, in-frame deletions of 46 and 84 amino acids. Two X-ray-resistant revertants of XR-V15B expressed two Ku86 transcripts, one with and one without the deletion, suggesting that reversion occurred by activation of a silent wild-type allele. Transfection of fulllength cDNA encoding hamster Ku86 into XR-V15B cells resulted in a complete rescue of DNA-end-binding (DEB) activity and Ku70 levels, suggesting that Ku86 stabilizes the Ku70 polypeptide. In addition, cells expressing wild-type levels of DEB activity were fully rescued for X-ray resistance and V(D)J recombination, whereas cells expressing lower levels of DEB activity were only partially rescued. Thus, Ku is an essential component of the pathway(s) utilized for the resolution of DNA double-strand breaks induced by either X rays or V(D)J recombination, and mutations in the Ku86 gene are responsible for the phenotype of group 5 cells.All cells possess a mechanism for repairing DNA doublestrand breaks (DSBs) produced by ionizing radiation. Cells of the immune system must also resolve DNA DSBs produced by V(D)J recombination of the immunoglobulin and T-cell receptor genes during development of B and T cells (reviewed in reference 28). In fact, the biochemical pathways for DSB repair and V(D)J recombination have a number of common factors. Evidence of this was first described for the severe combined immune deficient (scid) mouse, which is hypersensitive to ionizing radiation because of defective DSB repair (1, 12, 18) and immune deficient because of defective V(D)J recombination (29). Subsequently, other X-ray-sensitive rodent cell lines defective in DSB repair were also found to have impaired V(D)J recombination (17,27,34,45,47). These cell lines fall into four complementation groups, 4, 5, 6, and 7, with group 7 corresponding to the scid defect (43, 52). The human genes capable of rescuing these mutants are designated XRCC4, XRCC5, XRCC6, and XRCC7, respectively (XRCC denotes X ray cross-complementing) (for a review, see reference 51).Recently, it has been shown that the Ku protein is involved in both DSB repair and V(D)J recombination. Ku is an abundant nuclear protein identified originally as an autoantigen recognized by sera from patients with autoimmune diseases, including scleroderma-polymyositis overlap syndrome and systemic lupus erythematosus (31