The G protein-coupled receptor 54 (GPR54) and its endogenous ligand, kisspeptin, are essential for activation and regulation of the hypothalamic-pituitary-gonadal axis. Analysis of RNA extracts from individually identified hypothalamic GnRH neurons with primers for GnRH, KiSS-1, and GPR54 revealed expression of all three gene products. Also, constitutive and GnRH agonist-induced bioluminescence resonance energy transfer (BRET²) between Renilla luciferase (Rluc) tagged GnRH-R and GPR54 tagged with green fluorescent protein (GFP²), expressed in HEK-293 cells, revealed hetero-oligomerization of the two receptors. Whole cell patch-clamp recordings from identified GnRH neurons showed initial depolarizing effects of kisspeptin on membrane potential, followed by increased action potential firing. In perifusion studies, treatment of GT1–7 neuronal cells with kisspeptin-10 increased GnRH peak-amplitude and duration. The production and secretion of kisspeptin in cultured hypothalamic neurons and GT1–7 cells was detected by a specific radioimmunoassy, and was significantly reduced by treatment with GnRH. The expression of kisspeptin and GPR54 mRNAs in identified hypothalamic GnRH neurons, as well as kisspeptin secretion, indicate that kisspeptins may act as paracrine and/or autocrine regulators of the GnRH neuron. Stimulation of GnRH secretion by kisspeptin, and the opposing effects of GnRH on kisspeptin secretion, indicates that GnRH receptor/GnRH and GPR54/kisspeptin autoregulatory systems are integrated by negative feedback to regulate GnRH and kisspeptin secretion from GnRH neurons.