The inhibitory effect of various gestagens upon the pregnenolone 3 beta-ol-dehydrogenase-delta5-4-isomerase system in 10,000 X g supernatants of human corpora lutea of the menstrual cycle has been investigated in in vitro experiments. The Km value for pregnenolone was 1.3 X 10(-6) M. All gestations reduced the rate of conversion of pregnenolone to progesterone. As compared with controls, average relative rates of reaction with additives were as follows: progesterone, 47%; 17-hydroxyprogesterone, 64% norethisterone, 33%; chlormadinone acetate, 47% medroxyprogesterone acetate, 53%; dydrogesterone, 62%; and allylestrenol, 74%. The data substantiate that natural or synthetic gestagens, at least in vitro, inhibit the conversion of pregnenolone to progesterone by human corpora lutea and that natural progesterone itself is a potent inhibitor which appears to acut noncompetitively (K1 =2.2 X 10(-5) M).