The XO sex chromosome constitution has been found in both sexes of the mole-vole (Ellobius lutescens) belonging to the rodent family Microtinae. This enigmatic species has apparently been enduring a 50% zygotic lethality. The current serological study revealed the presence in XO males and the absence from XO females of H-Y (histocompatibility Y) antigen. In all the mammalian species studied thus far, the expression of H-Y antigen strictly coincided with the presence of testicular tissue and not necessarily with the presence of the Y chromosome. The testis-organizing function of the H-Y gene appears to have been confirmed. In the mole-vole, X linkage of the testis-organizing H-Y gene is favored over its autosomal inheritance. Only X linkage of the H-Y gene creates a compelling evolutionary need to change the female sex chromosome constitution from XX to XO, and to abandon the dosage compensation by an X inactivation mechanism, so that the nonproductive XH-YX zygote can be eliminated as an embryonic lethal. With regard to the electrophoretic mobilities of three X-linked marker enzymes, however, a genetic difference between the male-specific XH-Y and the female-specific X was not detected. This might reflect a relatively recent speciation.