The inhibition of the conversion of pregnenolone to progesterone by endogenous substances previously observed in rat and human placentas in vitro was investigated in placentas of 3 additional mammalian species. 20.alpha.-Hydroxy-pregn-4-ene-3-one, produced in vivo by reduction of the 20-ketone group of progesterone, inhibited this conversion in placental homogenates from cow, goat and rhesus monkey. The apparent Ki''s ranged from 0.5-3.4 .mu.M. In contrast, in vitro progesterone synthesis in corpora lutea of pregnancy from rabbit, rat and a 12-wk human pregnancy was little affected by 20.alpha.-hydroxy-pregn-4-ene-3-one. Cortisone also inhibited progesterone synthesis by placental homogenates from man, cow, goat and monkey. Rat placentas were not studied. Measurements on related C-21 steroids revealed considerable structural specificity, with cortisone the most effective inhibitor tested in this group of compounds. Prostaglandins A1, A2, B2, E1, E2 and F2 .alpha. had no apparent effect on the reaction. The progressive increase in specific activity of placental 3.beta.-hydroxy-.DELTA.5-steroid dehydrogenase on dilution previously described for man and rat was also apparent in cow, goat and rhesus monkey, supporting earlier suggestions of the existence of another endogenous inhibitor. Hypotheses are presented that both 20.alpha.-hydroxypregn-4-ene-3-one and the 2nd inhibitory substance are concerned with control and/or maintenance of progesterone levels during pregnancy, while cortisone is involved in reduction of these levels at the time of parturition.