Mammalian preovulatory follicles produce primarily estradiol and androgens before the preovulatory gonadotropin surge. In cattle, the LH/FSH surge triggers a rapid decrease in estradiol and androgen production and a dramatic increase in progesterone and oxytocin biosynthesis. It is unclear how changes in gonadotropin concentrations in vivo regulate this follicular/luteal phase shift in hormone production. To address this question, theca interna and granulosa cells were isolated from preovulatory follicles of Holstein heifers approximately 24 h before the expected time of the LH/FSH surge and cultured in defined medium containing insulin (1 microgram/ml), with varying doses of LH or FSH (0, 1, 2, 4, 8, 16, 32, 64, or 128 ng/ml). Media were collected and replaced every 24 h for 3 days, and assayed for androstenedione and progesterone in theca interna cultures, and for estradiol, progesterone and oxytocin in granulosa cell cultures. After the first day of culture (24-72 h), only very low doses of LH (2 or 4 ng/ml) enhanced (p < 0.05) androstenedione secretion by theca interna cultures above control levels, whereas progesterone secretion was increased by a wide range of LH concentrations (p < 0.05), with maximal progesterone secretion at high doses of LH. Likewise, after the first day of culture (24-72 h), estradiol secretion by granulosa cells was stimulated only by low doses of FSH and was inhibited at higher concentrations relative to control cultures (p < 0.05), whereas the production of oxytocin and progesterone was enhanced maximally by high concentrations of FSH (p < 0.05).(ABSTRACT TRUNCATED AT 250 WORDS)