Kisspeptin is the peptide product of the KiSS‐1 gene and the endogenous agonist for the GPR54 receptor. Recent evidence suggests the kisspeptin/GPR54 system is a key regulator of the reproductive system. We examined the effect of intracerebroventricular (i.c.v.) and peripheral administration of the active kisspeptin fragment, kisspeptin‐10, on circulating gonadotrophins and total testosterone levels in adult male rats. The effect of kisspeptin‐10 in vitro on the release of hypothalamic peptides from hypothalamic explants and gonadotrophins from anterior pituitary fragments was also determined. The i.c.v. administration of kisspeptin‐10 dose‐dependently increased plasma luteinizing hormone (LH) and increased plasma follicle stimulating hormone (FSH) and total testosterone at 60 min postinjection. In a separate study investigating the time course of this response, i.c.v. administered kisspeptin‐10 (3 nmol) significantly increased plasma LH at 10, 20 and 60 min, FSH at 60 min and total testosterone at 20 and 60 min postinjection. Kisspeptin‐10 stimulated the release of luteinizing hormone‐releasing hormone (LHRH) from in vitro hypothalamic explants. Peripheral administration of kisspeptin‐10 increased plasma LH, FSH and total testosterone. However, doses of 100–1000 nM kisspeptin‐10 did not influence LH or FSH release from pituitary fragments in vitro. Kisspeptin therefore potently stimulates the hypothalamic‐pituitary‐gonadal axis. These effects are likely to be mediated via the hypothalamic LHRH system.