The output of progesterone by explants of chorion incubated without exogenous precursor was increased in the presence of 20α-dihydroprogesterone (20α-diHP). We have now examined the nature of the 20α-diHP effect. Explants of chorion were incubated with [3H]progesterone and 3 μM non-radiolabelled 20α-diHP to examine the effect of 20α-diHP on progesterone metabolism. Significantly more radioactivity remained as unmetabolized [3H]progesterone compared to chorion incubated in the absence of non-radiolabelled 20α-diHP. There was also a decrease in the output of [3H]-20α-diHP by the explants but no change in the output of a second metabolite of progesterone [3H]-5α-pregnanedione. When explants were incubated with 20α-diHP in the presence of the 3β-hydroxysteroid dehydrogenase inhibitor, trilostane, the output of progesterone was significantly greater than with explants incubated with trilostane but without addition of 20α-diHP. Radiochemically pure [3H]progesterone was produced by explants of chorion incubated with [3H]-20α-diHP. We conclude that 20α-diHP may serve as substrate for chorionic progesterone production through 20α-hydroxysteroid dehydrogenase. 20α-diHP, formed from progesterone, may also exert product feedback inhibition on this enzyme.