In a previous in vitro study we found that androgens and estradiol (E2) suppress progesterone (P4) production by the granulosa cells isolated from the largest follicle of the domestic hen in a dose-dependent manner. The presence of an aromatase inhibitor did not block the inhibitory action of androgens. The addition of androgen plus E2 to the granulosa cells had an additive effect on suppressing P4 secretion. The aim of this study was to determine the loci in the steroid biosynthetic pathway where androgens and E2 inhibit P4 production by the granulosa cells. Granulosa layers of the largest follicles removed from two or three hens 22 h before ovulation were pooled. Dispersed granulosa cells were incubated for 3 h in triplicate for each treatment, and pregnenolone (P5) and P4 secretion were measured in medium and cells. Experiments were replicated three or four times. Treatment of granulosa cells with cyanoketone (0-100 microM), an inhibitor of 3 beta-hydroxysteroid dehydrogenase, increased P5 production and decreased P4 production in a dose-dependent manner, with maximal production of P5 and suppression of P4 production at 10 microM cyanoketone. The addition of 25-hydroxycholesterol (25OHCh) or P5 (0-16 microM) caused a dose-related increase in basal and LH-stimulated steroid production. The maximal production of P5 or P4 was found at 8 microM 25OHCh or P5. Also, the effect of LH (0-100 ng) on granulosa cell steroidogenesis was examined with or without 8 microM 25OHCh or P5. The half-maximal and maximal doses for P5 or P4 production were 5 and 25 ng LH, respectively. Next, suppression of P5 production by androstenedione, testosterone, dihydrotestosterone, and E2 (each at 0-10 microM) was tested in the presence of 25OHCh plus cyanoketone with or without LH. We found a dose-dependent suppression of P5 production by androgens (1-10 microM), but not by E2. However, when we added the above steroids to granulosa cells in the presence of P5 with or without LH, only E2 (1 and 10 microM) caused a significant suppression of P4 production. Our results suggest that 1) androgens primarily act at the conversion site of cholesterol to P5 to suppress P4 production; and 2) E2 acts at the conversion site of P5 to P4 to suppress P4 production. We conclude that production of androgens and E2 by thecal cells may regulate P4 biosynthesis by granulosa cells in the domestic hen.