In placental mammals, the class II region of the major histocompatibility complex (Mhc) consists of several gene families which show orthologous relationships in the different species. As these families are not orthologous with the Mhc class II beta-chain-encoding gene families of birds, the different mammalian families must have diverged after the separation of birds and mammals approximately 250 Mya but before the radiation of placental mammals (60-80 Mya). To obtain further information about the origin of the class II genes in mammals, we studied the beta-chain-encoding genes of the wallaby as a representative of marsupials, which split from placental mammals approximately 125 Mya. Three beta-chain-encoding genes were isolated from a red-necked wallaby (Macropus rufogriseus) cDNA library by using a chimpanzee DRB probe, and their nucleotide sequences were determined. The genes are not orthologous to any of the genes in mammals studied thus far but belong to two new families which we designated Maru-DAB and Maru-DBB. One of the three sequences (DAB2) seems to be derived from a transcribed pseudogene; it lacks the codons specifying the first 51 amino acid residues of the beta 2 domain. The fact that the DAB and DBB families have thus far not been found in placental mammals and that none of the DOB, DPB, DQB, or DRB genes seems to be expressed in the one representative marsupial species can be interpreted as suggesting that class II gene families of eutherian and metatherian mammals evolved from different ancestral genes.