We report the cloning and mapping of a gene (PDHA) for the pyruvate dehydrogenase E1 alpha subunit in marsupials. In situ hybridization and Southern blot analysis show that PDHA is autosomal in marsupials, mapping to chromosome 3q in Sminthopsis macroura and 5p in Macropus eugenii. Since these locations represent a region that was translocated to the p arm of the human X chromosome following marsupial/eutherian divergence, we suggest that the marsupial PDHA gene is homologous to PDHA1, the somatic eutherian isoform located on human Xp and mouse X. Only one copy of PDHA is found in marsupials, whereas a second, testis-specific, intronless form is observed in eutherian mammals. We also suggest that translocation of PDHA to the eutherian X chromosome, which is inactivated during spermatogenesis, led to the evolution of a second testis-specific locus by retroposition to an autosome.