The positive feedback effect of estradiol on the hypothalamic-pituitary axis is believed to initiate the periovulatory sequence of circulating hormone changes. This study was designed to investigate the temporal relationships between elevated serum estradiol levels and the luteinizing hormone (LH) surge. Six amenorrheic women received a single intramuscular injection of estradiol benzoate (EB) in a dose of 50 μg per kilogram, and subsequent blood levels of estradiol (E2), LH, and FSH were followed at six-hour intervals. All patients demonstrated a positive gonadotropin response, mainly LH, at a varaible time after EB administration. There was a latent period of 12 to 40 hours after the first estrogen peak before the increase in gonadotropin secretion. Augmented gonadotropin release appears to result from falling levels of circulating estradiol and elevated levels of estradiol appear to inhibit release of LH from the pituitary gland.