The positive feedback effects of estradiol (E2) and progesterone (P) on LH and FSH release were studied under novel experimental conditions in three women of reproductive age who had undergone oophorectomy and received uninterupted E2 replacement by subdermal implants. Basal serum E2 levels were in the midfollicular phase range, while LH and FSH levels were normal or slightly elevated. Each volunteer underwent seven experiments at 2- to 4-week intervals, receiving im injections of increasing amounts of estradiol benzoate (E2B) alone and in combination with P. The time and dose of P (administered via P-impregnated polysiloxane intravaginal rings) were varied. In two of the seven experiments, P was given without E2B injections. In all three subjects, increasing serum E2 levels mimicking the preovulatory E2 peak were followed by a surge of LH but not of FSH. However, when serum P levels rose after an increase in serum E2 concentrations had occurred, the LH surge occurred earlier and was accompanied by an FSH peak. When serum P levels rose before serum E2 concentrations had risen or when P levels increased without a rise in serum E2, neither a serum LH nor FSH peak was observed. When administered concomitantly, E2B and P suppressed FSH but not LH levels, while P alone did not affect serum LH or FSH concentrations. These data indicate that an acute rise in serum E2 is a necessary condition for the midcycle LH and FSH surges, that P facilitates or blocks the positive feedback response of gonadotropin release in a time-dependent manner, and that P is required for the preovulatory FSH peak.