Our recent study indicates that a brief infusion (20 min) of estradiol (E2) benzoate (EB) into the stalk-median eminence (S-ME) stimulates GnRH release with a latency of approximately 10 minutes. In contrast to the effect induced by a brief infusion of EB, it has previously been shown that systemic EB administration suppresses release of GnRH, kisspeptin, and LH with a latency of several hours, which is known as the negative feedback action of E2. We speculated that the differential results by these 2 modes of EB administration are due to the length of E2 exposure. Therefore, in the present study, the effects of EB infusion for periods of 20 minutes, 4 hours, or 7 hours into the S-ME of ovariectomized female monkeys on the release of GnRH and kisspeptin were examined using a microdialysis method. To assess the effects of the EB infusion on LH release, serum samples were also collected. The results show that similar to the results with 20-minute infusion, both 4- and 7-hour infusions of EB consistently stimulated release of GnRH and kisspeptin from the S-ME accompanied by LH release in the general circulation. In contrast, sc injection of EB suppressed all 3 hormones (GnRH, kisspeptin, and LH) measured. It is concluded that regardless of the exposure period, direct E2 action on GnRH and kisspeptin neurons in the S-ME, where their neuroterminals are present, is stimulatory, and the E2-negative feedback effects do not occur at the S-ME level.