Newly discovered kisspeptin (= metastin), encoded by Kiss1/KISS1 gene, is considered as a major gatekeeper of puberty through the regulation of gonadotropin-releasing hormone (GnRH). In the present study, we cloned a novel kisspeptin gene (kiss2) in the zebrafish Danio rerio and the medaka Oryzias latipes, which encodes a sequence of 125- and 115-amino acids respectively and its core sequence (FNLNPFGLRF = F-F form) is different from the previously characterized kiss1 (YNLNSFGLRY = Y-Y form). Our in silico data mining shows kiss1 and kiss2 are highly conserved across non-mammalian vertebrate species, and we have identified two putative kisspeptins in the platypus and three forms in the Xenopus. In the brain of zebrafish and medaka, in situ hybridization and laser capture microdissection coupled with real-time PCR showed kiss1 mRNA expression in the ventro-medial habenula and the periventricular hypothalamic nucleus. The kiss2 mRNA expression was observed in the posterior tuberal nucleus and the periventricular hypothalamic nucleus. Quantitative real-time PCR analysis during zebrafish development showed a significant increase in zebrafish kiss1, kiss2 (P<0.002), gnrh2 and gnrh3 (P<0.001) mRNA levels at the start of the pubertal phase and remained high in adulthood. In sexually mature female zebrafish, Kiss2 but not Kiss1 administration significantly increased follicle stimulating hormone beta (2.7-fold, P<0.05) and luteinizing hormone beta (8-fold, P<0.01) mRNA levels in the pituitary. These results suggest that the habenular Kiss1 and the hypothalamic Kiss2 are potential regulators of reproduction including puberty, and that Kiss2 is the predominant regulator of gonadotropin synthesis in fish.