The enzyme 20α-hydroxysteroid dehydrogenase(20α-HSD) plays a key role in the conversion of progesterone to the biological inactive steroid, 20α-hydroxypregn-4-en-3-one(20α-dihydroprogesterone; 20α-OHP). This study was carried out to investigate the activity of 20α-HSD in the rat ovary during pregnancy. Wistar Immamichi rats were impragnated by mating with the same strain of male rats. Concentrations of progesterone in the serum during pregnancy were higher from Day 12 to 18 than that of early pregnancy but dramatically decreased on day 21. Concentration of 20α-OHP was similarly low from day 6 to 18 of pregnancy and increased on Days 20 and 21. The total activity of ovarian cytosolic 20α-HSD was high on day 5 and 21, and decreased from day 10 to 18 of pregnancy. When ovarian 20α-HSD activity on day 5 of pregnancy was separated into HSD-1 and HSD-2 by DEAF-Toyopearl anion exchange chromatography, HSD-I activity was higher than HSD-2. This indicated that two isozymes of 20α-HSD(HSD-1 and HSD-21 may play an important role in the maintenance of pregnancy and the regulation of ovarian function.