The unusual immune response of urodele amphibians - low antibody heterogeneity, chronic skin graft rejection, weak mixed lymphocyte reactions - are much poorer than those of anuran amphibians, which are in turn poorer than those of mammals. Investigation of immunoglobulin (Ig) or T cell receptor (TCR) gene organization and diversification in urodeles has been stymied by the large size of the genome, which can be 20 times that of mammals. We present successful genomic Southern blotting of Ambystoma mexicanum (axolotl) DNA and formally demonstrate the presence of multiple V H and single Cμ genes in that urodele amphibian. A genomic DNA library was constructed from fractionated DNA, and two germline V H genes were isolated. Rearrangements to these V H gene segments, obtained from spleen RNA by the RT-PCR technique, were analyzed for heavy (H) chain junctional diversity and found to be even less variable than that in newborn mouse or Xenopus tadpoles. Only 29% of the CDR3 loop in the axolotl consisted of somatically generated sequences, compared to 44% in tadpole, 39% in newborn mice, and 57% in both adult mice and Xenopus. As the CDR3 loop is the most important portion of the Ig sequence for determining antibody combining site diversity, we suggest that the present finding is a molecular basis for the deficient urodele amphibian antibody responses documented in the literature.