Kisspeptin1 (product of the kiss1 gene) is the key neuropeptide that gates puberty and maintains fertility by regulating the gonadotropin-releasing hormone (GnRH) neuronal system in mammals. Inactivating mutations in kiss1 and the kisspeptin receptor (GPR54/KiSS1R) are associated with pubertal failure and infertility. Kiss2, a paralogous gene for kiss1, has been recently identified in several vertebrates including zebrafish. Using our transgenic zebrafish model system in which the GnRH3 promoter drives expression of emerald green fluorescent protein, we investigated the effects of kisspeptins on development of the GnRH neuronal system during embryogenesis and on electrical activity during adulthood. Quantitative PCR showed detectable levels of kiss1 and kiss2 mRNA by 1 day post fertilization, increasing throughout embryonic and larval development. Early treatment with kisspeptin1 or kisspeptin2 showed that both kisspeptins stimulated proliferation of trigeminal GnRH3 neurons located in the peripheral nervous system. However, only kisspeptin1, but not kisspeptin2, stimulated proliferation of the terminal nerve and hypothalamic populations of GnRH3 neurons in the central nervous system. Immunohistochemical analysis of synaptic vesicle protein 2 suggested that kisspeptin1, but not kisspeptin2, increased synaptic contacts along the terminal nerve-GnRH3 neuronal processes during embryogenesis. In intact brain of adult zebrafish, whole-cell patch clamp recordings of GnRH3 neurons from the preoptic area and hypothalamus revealed opposite effects of kisspeptin1 and kisspeptin2 on spontaneous action potential firing frequency and membrane potential. Kisspeptin1 increased spike frequency and depolarized membrane potential, whereas kisspeptin2 suppressed spike frequency and hyperpolarized membrane potential. We conclude that in zebrafish, kisspeptin1 is the primary stimulator of GnRH3 neuronal development in the embryo and an activator of stimulating hypophysiotropic neuron activities in the adult, while kisspeptin2 plays an additional role in stimulating embryonic development of the trigeminal neuronal population, but is an RFamide inhibitor of electrical activity of hypophysiotropic GnRH3 neurons in the adult.