Abstract The present study was designed to explore the steroidogenic responsiveness of ovine antral follicles of different sizes when cultured for varying time-periods with different doses of pregnenolone. Antral follicles of different sizes were isolated from sheep ovaries and cultured in medium 199 with or without pregnenolone in the presence or absence of FSH for 1, 6, 10, and 24 hours at 37 C. The levels of progesterone and estradiol in the spent medium were estimated. In the absence of pregnenolone, steroid production by the follicles did not increase significantly beyond 1 hour of culture. However, in the presence of pregnenolone there was a significant increase in progesterone production at 6, 10, and 24 hours of culture compared to controls. Estradiol levels were unaffected. Addition of FSH in combination with pregnenolone failed to increase progesterone or estradiol levels beyond that seen with pregnenolone alone. These data demonstrate that short-term incubation of follicles is sufficient for the secretion of steroids into the culture medium and supplementation of the culture medium with an immediate precursor is essential for optimal steroidogenesis in vitro.