The mammalian subclass Theria consists of infraclasses Metatheria (marsupials) and Eutheria ('placentals') which diverged from each other 120-150 million years before present (Myr BP). Both infraclasses have Y chromosome-dependent testis determination but direct molecular evidence linking the Metatherian and Eutherian Y chromosomes is lacking. Comparative analyses indicate that three mammalian genes have remained Y-linked for at least 80 Myr, since the divergence of the Eutherian orders from a common ancestor. These are Zfy, a gene encoding a transcription factor of the zinc-finger type; Sry, the putative primary testis-determining gene; and Ube1y (formerly Sby or A1s9Y-1), a candidate for the mouse spermatogenesis gene Spy, encoding a ubiquitin-activating enzyme E1 homologue. Although in marspials Zfy homologues are autosomal, a Y homologue of Sry has recently been isolated. We report here the identification of a functional marsupial Y-linked homologue of the murine Ube1y gene establishing that Metatherian and Eutherian Y chromosomes diverged from a common ancestor. This extreme conservation indicates that Ube1y plays a critical role in male development.