Kisspeptins (Kp) have recently emerged as master regulators of the reproductive axis and among the most potent elicitors of GnRH-gonadotropin secretion. Despite their paramount importance in reproductive physiology and their potential therapeutic implications, development of Kp antagonists has remained elusive, and only recently has the first compound with the ability to block Kp actions in vitro and in vivo, namely p234, been reported. However, previous in vivo studies all used acute central injections, whereas characterization of the effects of the antagonist after continuous or systemic administration, which poses pharmacological challenges, is still pending. We report herein a comprehensive series of analyses on the impact of continuous intracerebroventricular infusion of p234 on puberty onset and the preovulatory surge of gonadotropins in the female rat. In addition, the effects of systemic (ip) administration of a tagged p234-penetratin, with a predicted higher permeability at the blood-brain barrier, on Kp-10 induced gonadotropin secretion were evaluated. Central infusion of p234 to pubertal females delayed vaginal opening and decreased uterine and ovarian weights at the expected time of puberty, without affecting body weight. Likewise, chronic intracerebroventricular administration of p234 for 4 d prevented the preovulatory surges of LH and FSH. In addition, systemic (ip) administration of p234-penetratin significantly attenuated acute LH and FSH responses to Kp-10, either after intracerebroventricular or ip injection of Kp. Our data document the validity of p234 for antagonizing Kp actions in vivo and provide direct experimental evidence for the important role of Kp signaling in the key events of female reproduction, such as puberty onset and the preovulatory surge of gonadotropins.