Previously we have described the isolation and expression of RAG1 in trout to provide an initial understanding as to the tissues involved in V (D) J recombination of antigen receptors in this teleost. We now report that the recombination activating gene 2 (RAG2 ) of rainbow trout has now been cloned and characterized. The rainbow trout genomic RAG2 gene (1,601 BP) displays an average of 77% similarity at the amino acid level when compared to clones from other vertebrates species. The RAG genomic locus is similar to that found in other vertebrates. Both genes are convergently transcribed and share a 3′ untranslated region which is much shorter than that found in higher vertebrates. Northern blot analysis demonstrated strong expression of RAG2 in the thymus, with a weaker signal being detected in the pronephros. Using RT-PCR, the highest expression of both RAG1 and -2 were detected in the thymus and pronephros, with fainter signals being observed in the spleen, mesonephros and liver. Embryonic expression of both genes begins on day 10 post-fertilization. Additionally confocal microscopy was used to elucidate the location of putative pre-B cells in this teleost using an anti-heavy chain reagent. Taken together, these results provide insight regarding the sites and mechanisms of lymphopoiesis in teleosts as well as addressing questions concerning the origin(s) and evolution of our current immune system.