The neuropeptides kisspeptin, neurokinin B and dynorphin A are coexpressed within KNDy neurons that project from the hypothalamic arcuate nucleus to GnRH neurons. Neurokinin B (signaling through NK3R) and kisspeptin (signaling through GPR54) are potent stimulators of GnRH/LH secretion. In ewes, NK3R is abundantly expressed in the retrochiasmatic area (RCh) of the hypothalamus¹ and recent evidence from our group indicates that selective activation of RCh NK3R by senktide (NK3R agonist) triggers surge-like LH secretion in ovary-intact ewes¹, while their blockade by SB222200 (NK3R antagonist) suppresses the estradiol-induced LH surge². Furthermore, RCh senktide-induced LH secretion coincides with a significant increase in c-Fos (a marker of neuronal activation) expression within KNDy neurons, but not other kisspeptin neurons². The present study was designed to determine whether the surge-like LH secretion induced by intra-RCh administration of senktide is mediated by hypothalamic kisspeptin/GPR54 signaling. Ovary-intact ewes, with bilateral microimplants containing crystalline senktide inserted into the RCh, were subjected to frequent blood sampling during intracerebroventricular infusion of the GPR54 antagonist, Kp-271 (300 µg/h), or vehicle (saline; 120 µl/h). Neither vehicle nor Kp-271 had an effect on basal LH levels (mean ± SEM; 3.76 ± 0.31 ng/ml) prior to senktide administration. Senktide significantly increased LH levels (17.59 ± 1.46 ng/ml) 2 h following microimplant placement in ewes infused with vehicle. In contrast, the stimulatory effect of senktide on LH secretion was completely blocked (6.05 ± 0.62 ng/ml) by Kp-271 infusion. In the context of our previous findings², these data demonstrate that the stimulatory effect of RCh NK3R activation on LH secretion in ovary-intact ewes is dependent upon kisspeptin/GPR54 signaling, which likely originates from KNDy neurons. The discovery of this interaction provides further insight into the role of the RCh in the regulation of the LH surge in sheep.