Ovarian 20alpha-hydroxysteroid dehydrogenase (20alpha-HSD), which converts progesterone to a derivative devoid of biological activity, plays a crucial role in achieving the short estrous cycle in rats. Although 20alpha-HSD activity has also been demonstrated in the thymus, its molecular nature, function, and regulation of expression have yet to be determined. In the present study we investigated if 20alpha-HSD activity in the thymus originates in a transcript identical to that expressed in the ovary. RT-PCR analysis indicated the expression of 20alpha-HSD mRNA in rat thymus, and sequencing of the PCR product showed 100% identity to ovarian 20alpha-HSD cDNA. Immunohistochemical study using anti-rat ovarian 20alpha-HSD antibody demonstrated the expression of 20alpha-HSD protein in the thymus. The 20alpha-HSD-expressing cells in the thymus seemed to be some type of lymphocyte by their morphology. These results suggest that the same molecular species as ovarian 20a-HSD is expressed in thymic lymphocytes. Therefore, 20alpha-HSD may play a role in T-lymphocyte proliferation and differentiation processes.