The XR-1 Chinese hamster ovary cell line is impaired in DNA double-strand break repair (DSBR) and in ability to support V(D)J recombination of transiently introduced substrates. We now show that XR-1 cells support recombination-activating gene 1- and 2-mediated initiation of V(D)J recombination within a chromosomally integrated substrate, but are highly impaired in ability to complete the process by forming coding and recognition sequence joins. On this basis, we isolated a human cDNA sequence, termed XRCC4, whose expression confers normal V(D)J recombination ability and significant restoration of DSBR activity to XR-1, clearly demonstrating that this gene product is involved in both processes. The XRCC4 gene maps to the previously identified locus on human chromosome 5, is deleted in XR-1 cells, and encodes a ubiquitously expressed product unrelated to any described protein.