3β-Hydroxysteroid dehydrogenase (3β-HSD) activity in bovine cotyledons was much higher than in caruncles throughout the gestation period. The activity of this enzyme in cotyledons increased greatly to 150.6 ± 5.8 pmol min−1 mg−1 protein during the seventh month of gestation, reached a peak of 221.0 ± 34.9 pmol min−1 mg−1 protein during the eighth month, and decreased at parturition. Progesterone and 20α-hydroxyprogesterone concentrations in cotyledons also increased sharply to 2.69 ± 0.30 and 2.15 ± 0.42 ng mg−1 protein, respectively, during the seventh month of gestation, reaching peaks of 2.86 ± 0.47 and 2.51 ± 0.36 ng mg−1 protein, respectively, during the eighth month and decreasing at parturition, in a manner similar to the activity of 3β-hydroxysteroid dehydrogenase. The fluctuation of 17α-hydroxyprogesterone concentration in cotyledons was different from that of progesterone and 20α-hydroxyprogesterone. These findings indicate that the activity of 3β-hydroxysteroid dehydrogenase in the placenta is enhanced during the third trimester, and progesterone synthesized in the cotyledons is converted concurrently to 20α-hydroxyprogesterone before progesterone is transferred to the fetal blood.