The ability of cultured midpregnancy mouse ovarian cells to synthesize progesterone de novo and from oxogenous pregnenolone has been assessed. The conversion of pregnenolone to progesterone is almost completely blocked by cyanoketone, a known inhibitor of delta5,3beta-hydroxysteroid dehydrogenase (3beta-HSD) activity, but is unaffected by aminoglutethimide, which inhibits the cholesterol side-chain cleavage enzyme complex (desmolase). Since there is little metabolism of the formed progesterone, the ability of ovarian cells to convert exogenous pregnenolone to progesterone in vitro reflects the activity of 3beta-HSD in these cells. Cultured ovarian cells are also capable of endogenous progesterone production in the absence of added pregnenolone, although the absolute amount of progesterone produced is considerably less than that produced from exogenous pregnenolone. Since endogenous progesterone accumulation is almost completely blocked by the addition of aminoglutethimide to the culture medium, it is concluded that this response does represent de novo progesterone synthesis. Neither bovine luteinizing hormone (LH) nor human chorionic gonadotrophin (hCG) affects 3beta-HSD activity in cultured ovarian cells. The ability of the cells to secrete or to further metabolize the progesterone formed is also unaffected. However, both LH and hCG stimulate endogenous progesterone production within one hour of their addition to the culture medium. The stimulation, 2-10 fold in several experiments, can be maintained for at least six days of culture, and is not a result of an increase in the growth rate of the ovarian cells. As would be expected, the stimulation is blocked by the addition of aminoglutethimide to the culture medium. Taken together, these facts suggest that gonadotrophic hormones stimulate progesterone production by ovarian cells specifically by their action at steps prior to the conversion of pregnenolone to progesterone.