The recombinase activating genes (RAG1 and RAG2) encode nuclear proteins that directly mediate the mechanism V(D)J recombination process that occurs in T- and B-lymphocytes. The expression of RAG1 and RAG2 is required for the proper development of maturing lymphocytes. To identify evolutionary conserved regulatory regions adjacent to both genes we isolated and sequenced a cosmid clone containing 43kb of genomic DNA of the Japanese pufferfish, Fugu rubripes. Fugu has a haploid genome of 400Mb and contains the same number of genes as the genome of higher vertebrates. With low abundance of repetitive DNA, the genome of the pufferfish has shown to be ideal for comparative genomics. We found three complete genes, RAG1, RAG2 and ACS (a possible homologue of the plant 1-aminocyclopropane-carboxylate synthase gene). There is also the 5' exon of a prohormone convertase gene, possibly PACE4. The genetic structure of both RAG1 and RAG2 is identical to that found in other fish, but the size of the intergenic region is smaller in Fugu. Expression analysis by RT-PCR shows the presence of RAG transcripts in kidney of adult Fugu. The human ACS was identified in a cosmid assigned to chromosome 11p11, which is close to the location of the RAGs (11p12). This indicates conservation of linkage between human and pufferfish.