The X-linked aristaless gene,ARX, is essential for the development of the gonads, forebrain, olfactory bulb, pancreas, and skeletal muscle in mice and humans. Mutations cause neurological diseases, often accompanied by ambiguous genitalia. There are a disproportionately high number of testis and brain genes on the human and mouse X chromosomes. It is still unknown whether the X chromosome accrued these genes during its evolution or whether genes that find themselves on the X chromosome evolve such roles.ARXwas originally autosomal in mammals and remains so in marsupials, whereas in eutherian mammals it translocated to the X chromosome. In this study, we examined autosomalARXin tammars and compared it with the X-linkedArxin mice. We detectedARXmRNA in the neural cells of the forebrain, midbrain and hindbrain, and olfactory bulbs in developing tammars, consistent with the expression in mice.ARXwas detected by RT-PCR and mRNAin situhybridization in the developing tammar wallaby gonads of both sexes, suggestive of a role in sexual development as in mice. We also detectedARX/ArxmRNA in the adult testis in both tammars and mice, suggesting a potential novel role forARX/Arxin spermiogenesis.ARXtranscripts were predominantly observed in round spermatids.ArxmRNA localization distributions in the mouse adult testis suggest that it escaped meiotic sex chromosome inactivation during spermatogenesis. Our findings suggest thatARXin the therian mammal ancestor already played a role in male reproduction before it was recruited to the X chromosome in eutherians.