Kisspeptin (KP) and KP-1 receptor (KISS1R) have emerged as important upstream regulators in the control of puberty. However, how developmental changes in KP-KISS1R contribute to the pubertal increase in GnRH release still remains elusive. In this study, we examined the effects of the KP agonist, human KP-10 (hKP-10), and the KP antagonist, peptide 234, on in vivo GnRH release in prepubertal and pubertal ovarian-intact female rhesus monkeys using a microdialysis method. We found that direct infusion of hKP-10 into the medial basal hypothalamus and stalk-median eminence region stimulated GnRH release in a dose-responsive manner, whereas infusion of peptide 234 suppressed GnRH release in both developmental stages. Because ovarian steroid feedback on GnRH release becomes prominent after the initiation of puberty in primates, we further examined whether ovarian steroids modify the GnRH response to hKP-10. Results demonstrate that the hKP-10-induced stimulation of GnRH release was eliminated by ovariectomy in pubertal, but not prepubertal, monkeys. Furthermore, replacement of estradiol into ovariectomized pubertal monkeys resulted in a partial recovery of the hKP-10-induced GnRH release. Collectively, these results suggest that a KISS1R-mediated mechanism, in addition to the pubertal increase in KP-54 release we previously reported, contributes to the pubertal increase in GnRH release and that there is a switch from an ovarian steroid-independent to -dependent mechanism in the response of GnRH to KP.