Abstract Somatic recombination of immunoglobulin or T cell antigen receptor genes occurs through recombination activating gene product (RAG)-mediated cleavage at recombination signal sequences (RSS) and subsequently, the broken DNA ends are repaired by non-homologue end joining (NHEJ) enzymes. Here, we show that RAG1, RAG2, and many NHEJ factors in B lineage cells are associated with the nuclear matrix. The core RAG1 and RAG2 proteins have their own nuclear matrix targeting regions. RAG-mediated double stranded DNA breaks at the Jκ4 RSS can be readily detected in the nuclear matrix fraction in Gleevec treated Abelson transformed murine B cells or mouse bone marrow cells, indicating that the recombination reaction is ongoing on the nuclear matrix. In the HEK 293 cell-based recombination system, artificial recombination substrates are recruited to the nuclear matrix for recombination. Moreover, nuclear matrix proteins purified from 293 cells expressing the core RAG proteins or human B lineage cells expressing endogenous RAG proteins support cleavage of RSS substrates in vitro. Based on these results, we propose that the V(D)J recombination machinery is associated with the nuclear matrix.