We have previously demonstrated that a constant intravenous infusion of kisspeptin (Kp) for 48 h in anestrous ewes induces a preovulatory luteinizing hormone (LH) surge followed by ovulation in approximately 75% of animals. The mechanisms underlying this effect are unknown. In this study, we investigated whether Kp-induced preovulatory LH surges in anestrous ewes were the result of the general activation of the whole gonadotropic axis or of the direct activation of central GnRH neurons required for the GnRH/LH surge. In the first experiment, a constant iv infusion of ovine kisspeptin 10 (Kp; 15.2 nmol/h) was given to 11 seasonally acyclic ewes over 43 h. Blood samples were taken every 10 min for 15 h, starting 5 h before the infusion, and then hourly until the end of the infusion. We found that the infusion of Kp induced a well-synchronized LH surge (around 22 h after the start of the Kp infusion) in 82% of the animals. In all ewes with an LH surge, there was an immediate but transient increase in the plasma concentrations of LH, follicle-stimulating hormone (FSH), and growth hormone (GH) at the start of the Kp infusion. Mean (+/- SEM) concentrations for the 5-h periods preceding and following the start of the Kp infusion were, respectively, 0.33 +/- 0.09 vs 2.83 +/- 0.49 ng/mL (P = 0.004) for LH, 0.43 +/- 0.05 vs 0.55 +/- 0.03 ng/mL (P = 0.015) for FSH, and 9.34 +/- 1.01 vs 11.51 +/- 0.92 ng/mL (P = 0.004) for GH. In the first experiment, surges of LH were observed only in ewes that also had a sustained rise in plasma concentrations of estradiol (E(2)) in response to Kp. Therefore, a second experiment was undertaken to determine the minimum duration of Kp infusion necessary to induce such a pronounced and prolonged increase in plasma E(2) concentration. Kisspeptin (15.2 nmol/h) was infused for 6, 12, or 24 h in seasonally acyclic ewes (N = 8), and blood samples were collected hourly for 28 h (beginning 5 h before the start of infusion), then every 2 h for the following 22 h. Kisspeptin infused for 24 h induced LH surges in 75% of animals, and this percentage decreased with the duration of the infusion (12 h = 50%; 6 h = 12.5%). The plasma concentration of E(2) was greater in ewes with an LH surge compared to those without LH surges; mean (+/- SEM) concentrations for the 5-h period following the Kp infusion were, respectively, 2.23 +/- 0.16 vs 1.27 +/- 0.13 pg/mL (P < 0.001). Collectively, our results strongly suggest that the systemic delivery of Kp induced LH surges by activating E(2)-positive feedback on gonadotropin secretion in acyclic ewes.