Kisspeptin-54 is the peptide product of the KiSS-1 gene and an endogenous agonist of the GPR54 receptor. KiSS-1 was initially discovered as a metastasis suppressor gene, but recent studies demonstrate that the kisspeptin/GPR54 system is a key regulator of the reproductive system. Disrupted GPR54 signalling causes hypogonadotrophic hypogonadism in rodents and man. Intracerebroventricular or peripheral administration of kisspeptin potently stimulates the hypothalamic-pituitary-gonadal (HPG) axis via the hypothalamic gonadotrophin-releasing hormone system. We have investigated the effect of injection of kisspeptin-54 into discrete hypothalamic regions on the HPG axis. To construct a dose-response curve for the effects of intrahypothalamic kisspeptin administration, adult male Wistar rats were cannulated into the medial preoptic area (MPOA) at the level of the organum vasculosum laminae terminalis (OVLT). Kisspeptin-54 was injected into the MPOA at doses of 0.01, 0.1, 1, 10 and 100 pmol. At 60 min following injection of 1, 10 or 100 pmol kisspeptin-54, plasma luteinising hormone (LH) and total testosterone levels were significantly increased. Adult male Wistar rats were then cannulated into the rostral preoptic area at the level of the OVLT (RPOA), the MPOA, the paraventricular (PVN), dorsomedial (DMN) and arcuate hypothalamic nuclei, and the lateral hypothalamic area. A dose of 1 pmol kisspeptin-54 was administered into all areas. The circulating levels of LH and total testosterone were significantly increased 60 min postinjection of kisspeptin-54 into the RPOA, MPOA, PVN and arcuate nucleus. Our results suggest that kisspeptin may mediate its effects on the HPG axis via these regions of the hypothalamus.