The G protein-coupled receptor GPR54, and its peptide ligand kisspeptin, are crucial for the induction and maintenance of mammalian reproductive function. GPR54 is expressed by gonadotropin-releasing hormone (GnRH) neurons and is directly activated by kisspeptin to stimulate GnRH release. We hypothesized that kisspeptin may be able to act at the GnRH nerve terminals located in the mediobasal hypothalamus (MBH) region. To test this hypothesis we used organotypic culture of MBH explants challenged with kisspeptin followed by radioimmunoassay to detect GnRH released into the cultured medium. Kisspeptin stimulation for 1 hour induced GnRH release from wild-type male MBH in a dose-dependent manner whereas this did not occur in MBH explants isolated from Gpr54 null mice. Continuous kisspeptin stimulation caused a sustained GnRH release for four hours followed by a fall of GnRH release suggesting a desensitization of GPR54 activity. Tetrodotoxin did not alter the kisspeptin-induced GnRH release indicating that kisspeptin can act directly at the GnRH nerve terminals. To localize Gpr54 expression within the MBH, we used transgenic mice, where Gpr54 expression is tagged with an IRES-LacZ reporter gene and can be visualized by a -galactosidase staining. Gpr54 expression was detected outside of the median eminence, in the pars tuberalis. In conclusion, our results provide evidence for a potent stimulating effect of kisspeptin at GnRH nerve terminals in the MBH of the mouse. This study suggests a new point at which kisspeptin can act on GnRH neurons.