Lymphocyte antigen receptor variable regions are encoded by gene segments that are assembled by the site-specific variable (diversity) joining (V(D)J) recombination process. We have assayed the V-3 Chinese hamster ovary cell line, which has a double-strand DNA break repair (dsbr) defect, for the ability to carry out V(D)J recombination following transfection of constructs that encode the RAG-1 and RAG-2 proteins necessary to confer V(D)J recombination activity to non-lymphoid cells. The V-3 cells had substantially impaired ability to undergo V(D)J recombination of transiently introduced test substrates. Although these cells can initiate V(D)J recombination by introducing endonucleolytic scissions at the junctions of V(D)J recombination signal sequences and the flanking coding sequences, they have a greatly impaired ability to rejoin the coding sequences. Detailed characterization of attempted coding joins recovered from these cells indicated that the V(D)J recombination defect in V-3 is similar in phenotype to the murine severe combined immunodeficient (scid) defect and quite distinct from that found in other Chinese hamster ovary dsbr mutant cell lines. Somatic cell complementation analyses between homozygous scid mutant fibroblasts and V-3 cells confirmed that these mutations fall into the same genetic complementation group.