The objective of the present study was to determine the interactions among bST, insulin, and gonadotropins on steroid production by granulosa and thecal cells from bovine follicles. Basal production of estradiol by granulosa cells from small (1 to 5 mm) and large (> or = 8 mm) follicles (expressed as picograms of estradiol per 10(5) cells per 24 h) was not affected by 50 or 300 ng/ml of bST, but 300 ng/ml of bST inhibited estradiol production that was induced by FSH plus insulin in cells from small and large follicles. Progesterone production and proliferation by granulosa cells from large follicles were not affected by 3 to 100 ng/ml of bST. In cultures of thecal cells that exhibited a > 3-fold increase in androstenedione production induced by LH, 3 to 30 ng/ml of bST further increased androstenedione production by 29 to 42%, but cell proliferation and progesterone production were unaffected by bST. In cultures of thecal cells that exhibited a < 2-fold increase in androstenedione production induced by LH, 3 to 30 ng/ml of bST inhibited androstenedione production by 32 to 33% and inhibited cell proliferation by 9 to 13%, but progesterone was unaffected by bST. In summary, only pharmacologic doses of bST inhibited estradiol production by granulosa cells, but physiologic doses of bST altered androstenedione production by thecal cells, which indicated that bST might not have an important role in granulosa cell function but might play a role in thecal cell function in cattle.