The RAG-1 (recombination activating gene-l) genomic locus, which activates V (O) J recombination when introduced into NIH 3T3 fibroblasts, was isolated by serial genomic transfections of oligonucleotide-tagged DNA. A genomic walk spanning 55 kb yielded a RAG-1 genomic probe that detects a single 6.6-7.0 kb mRNA species in transfectants and pre-B and pre-T cells. RAG-1 genomic and cDNA clones were biologically active when introduced into NIH 3T3 cells. Nucleotide sequencing of human and mouse RAG-1 cONA clones predicts 119 kd proteins of 1043 and 1040 amino acids, respectively, with 90% sequence identity. RAG-1 has been conserved between species that carry out V (O) J recombination, and its pattern of expression correlates exactly with the pattern of expfession of V (O) J recombinase activity. RAG-1 may activate V (O) J recombination indirectly, or it may encode the V (O) J recombinase itself.