Human chorion can synthesize and metabolize progesterone, and changes in progesterone synthesis by chorion at term might be important in the processes leading to parturition. We examined whether other steroids present within the maternal compartment and amniotic fluid during late pregnancy influence progesterone output by explants of chorion. We also sought differences in steroid effects on progesterone output in association with labor. Explants were prepared from chorion collected after the spontaneous onset of labor and vaginal delivery and chorion collected after cesarean section without active labor. To study the short-term effects of steroids on progesterone output by chorion, explants were incubated for 4 h with 3 μM pregnenolone and 3 μM of a potential interacting steroid. Other explants were preincubated for 24 h with steroid, then rinsed and incubated for 4 h with 3 μM pregnenolone and 3 μM of the same steroid as during preincubation. Under these conditions, dehydroepiandrosterone and androstenedione inhibited progesterone output by explants of chorion obtained at spontaneous labor and at cesarean section. Testosterone also inhibited progesterone output, but only in cesarean section chorion. If explants were preincubated for 24 h with steroid and then rinsed and incubated for 4 h with pregnenolone only, progesterone synthesis returned to control values. This finding indicates that the mechanism of action of these inhibitory steroids is likely through an effect on 3β-HSD activity and not due to a change in the rate of enzyme synthesis. We also noted apparent stimulatory effects of steroids. When explants were preincubated with pregnenolone sulfate and then incubated for 4 h with 3 μM pregnenolone only or with 3 μM pregnenolone +3 μM pregnenolone sulfate, there was an increase in media progesterone concentrations. To examine the effects of steroids on basal progesterone synthesis, we incubated explants for 24 h with 3 μM steroid without addition of exogenous pregnenolone. 5α-dihydrotestosterone in spontaneous vaginal chorion and 5α-pregnanediol in cesarean section chorion caused an increase in media progesterone concentrations. Progesterone concentrations increased during 24-hour incubations with 20α-dihydroprogesterone and pregnenolone sulfate; the effect of pregnenolone sulfate was greater in chorion collected at spontaneous labor than at elective cesarean section. Addition of pregnenolone sulfate with pregnenolone during 24-hour incubations and during the 2nd incubation of 4 h further increased progesterone accumulation in the media. We conclude that several steroids can modify net progesterone output by explants of chorion in the presence of endogenous or exogenous precursors and that the steroid effects vary with the type of delivery.