BACKGROUND Studies in humans and animals indicate that estrogen negative feedback occurs at the level of the hypothalamus, but it is unclear whether estrogen also exerts an inhibitory effect directly at the pituitary. OBJECTIVE The aim of the study was to determine whether estrogen has a direct negative feedback effect at the pituitary and whether this varies with aging. METHODS A GnRH antagonist and graded doses of GnRH were used to isolate pituitary responsiveness before and after estrogen administration in Clinical Research Center studies at an academic medical center. METHODS Subjects were healthy postmenopausal women aged 48-56 yr (n = 8) or 70-75 yr (n= 8). METHODS A suppressive dose of the NAL-GLU GnRH antagonist was administered, followed by graded doses of GnRH before and after 1 month of estrogen administration. RESULTS LH and FSH responses to GnRH decreased after estrogen administration (P = 0.01 and P = 0.0001, respectively). The ratio of FSH to LH amplitudes decreased in response to estrogen (P = 0.04) indicating a greater sensitivity of FSH than LH to inhibition by estrogen. The inhibitory effect of estrogen on FSH was attenuated with aging (P = 0.02), but was maintained for LH (P = 0.4). CONCLUSIONS Studies that control for endogenous GnRH and estradiol demonstrate a direct pituitary site of estrogen negative feedback on LH and FSH responsiveness to GnRH in women. The effect of estrogen on FSH responsiveness is greater than on LH and is attenuated with aging. These studies indicate that estrogen negative feedback occurs directly at the pituitary and contributes to the differential regulation of FSH and LH secretion.