Recently, a novel physiologically active peptide, kisspeptin (metastin), has been reported to facilitate sexual maturation and ovulation by directly stimulating GnRH neurons in several mammalian species. In spite of its importance in the neuroendocrine regulation of reproduction, kisspeptin neurons have only been studied in mammals, and there has been no report on the kisspeptin or kisspeptin neuronal systems in non-mammalian vertebrates. We used medaka for the initial identification of KiSS-1 gene and the anatomical distribution of KiSS-1 mRNA expressing neurons (KiSS-1 neurons) in the brain of non-mammalian species. In situ hybridization for medaka KiSS-1 gene cloned here proved that two kisspeptin neuronal populations are localized in the hypothalamic nuclei, the nucleus posterioris periventricularis (NPPv) and the nucleus ventral tuberis (NVT). Furthermore, NVT KiSS-1 neurons were sexually dimorphic in number (male neurons > > female neurons) under the breeding conditions. We also found that the number of KiSS-1 neurons in the NVT but not that in the NPPv was positively regulated by ovarian estrogens. The fact that there were clear differences in the number of NVT KiSS-1 neurons between the fish under the breeding and non-breeding conditions strongly suggests that the steroid-sensitive changes in the KiSS-1 mRNA expression in the NVT occur physiologically, according to the changes in the reproductive state. From the present results, we conclude that the medaka KiSS-1 neuronal system is involved in the central regulation of reproductive functions, and, given many experimental advantages, the medaka brain may serve as a good model system to study its physiology.