Kisspeptin is a family of neuropeptides and the natural ligands of G protein-coupled receptor (GPR)-54. Kisspeptin/GPR-54 system is known to play a pivotal role in puberty onset and in the regulation of reproductive functions. To clarify the postnatal ontogeny of kisspeptin neurons in rat hypothalamus, we analyzed the expression patterns of kisspeptin mRNA from neonate to adult by in situ hybridization. In anteroventral periventricular nucleus (AVPV), kisspeptin mRNA were first detected at postnatal day (PND) 7 and postnatal week 3 in males and females, respectively, and the number of kisspeptin mRNA-expressing neurons increased during development in both sexes. In the arcuate nucleus (ARC), kisspeptin mRNA was present from PND3. In males, the number of kisspeptin mRNA-expressing neurons gradually increased during development. In females, the number of kisspeptin mRNA-expressing neurons in neonates was greater than in males; it significantly decreased at juvenile stages and then increased toward adulthood. These results indicate that the increase in kisspeptin mRNA expression in ARC across puberty might be involved in the onset of puberty. We also demonstrate that the kisspeptin mRNA-expressing neurons in ARC appear earlier than those in AVPV and show clear sex differences in their numbers during neonatal period.