The neuropeptide FF receptor antagonist 1-adamantanecarbonyl-Arg-Phe-NH2 trifluoroacetate salt (RF9) has been found to be a remarkably potent activator of gonadotropin secretion in mammals. However, the mechanism of RF9 action on the reproductive axis is unknown. Using acute brain slice electrophysiology in genetically modified mouse models, we have investigated the possibility that RF9 may activate GnRH neurons. In transgenic GnRH-GFP male and female mice, RF9 was found to exert potent, dose-dependent, stimulatory effects on the firing rate of approximately 70% of GnRH neurons. These effects occurred directly on GnRH neurons and were independent of fast amino acid transmission. To assess RF9's action as an neuropeptide FF receptor antagonist at the GnRH neuron, its ability to antagonize the inhibitory effects of RFamide-related peptide-3 on GnRH neuron firing was examined. RF9 exhibited variable ability to prevent the inhibitory effects of RFamide-related peptide-3 on GnRH neurons. Whole-cell recordings from GnRH neurons showed that RF9 generated an inward current in GnRH neurons reminiscent of that evoked by kisspeptin. We therefore examined RF9 actions in kisspeptin receptor knockout mice. RF9 was found to have no effects at all on GnRH neurons in GnRH-GFP;Kiss1r-null mice, although these cells exhibited normal intrinsic electrical properties and remained responsive to GABA and glutamate. This study reveals that RF9 directly activates GnRH neurons in the mouse and that this is dependent upon Kiss1r expression.