When contrasted with information available for placental mammals, very little is known about the development of immunocompetence in marsupials. Marsupials, however, provide interesting immunology problems, since most appear to be born at a stage of development much less mature than that of placental mammals. To further understand the marsupial immune system, the Ig repertoire of the short-tailed opossum, Monodelphis domestica, was characterized. The majority of the V(H) clones were isolated in an unbiased manner by screening a spleen cDNA phage library, using C region probes, or anchored PCR, using C region-specific primers paired with vector specific primers. Analysis of 54 unique V(H) sequences from this marsupial revealed the presence of two V(H) families in the expressed Ig repertoire. The larger family, which contributed the majority of the clones identified, appears to be derived from 10 to 12 germline V(H) segments. The second family of clones is derived from a single germline V(H). Both V(H) families are related to the group III sequences described in other vertebrates. Unusual codon bias differences between the two families may result in very different patterns of somatic mutation within the opossum Ig repertoire.