GnRH2 is a molecule conserved from fish to humans, suggesting its important functions. However, recent studies have shown that GnRH2 neurons project widely in the brain but not to the pituitary, which suggests their functions other than stimulation of gonadotropin secretion. In contrast to the wealth of knowledge in GnRH1 and GnRH3 neuronal systems, the GnRH2 neuronal system remains to be studied, and there has been no single cell approach so far, partly because of the lack of GnRH2 system in rodents. Here, we generated GnRH2-green fluorescent protein (GFP) transgenic medaka for the first single cell electrophysiological recording from GnRH2 neurons in vertebrates. Whole-cell and on-cell patch clamp analyses revealed their regular pacemaker activities that are intrinsic to the GnRH2 neurons. Pacemaker activities of GnRH2 neurons were not peculiar to medaka because dwarf gourami GnRH2 neurons also showed similar pacemaker activities. By comparing with spontaneous action currents from GFP-expressing GnRH1 and GnRH3 neurons in the adult transgenic medaka, which were already in our hands, we have demonstrated that GnRH2 neurons show pacemaker activity similar to nonhypophysiotropic GnRH3 neurons but not to hypophysiotropic GnRH1 neurons. Thus, by taking advantage of medaka brain, which has all three GnRH neuronal systems with different axonal projection patterns and thus different functions, we have gained insights into the close relationship between the pattern of spontaneous electrical activity and the functions of the three. Moreover, the three types of GnRH-GFP transgenic medaka will provide useful models for studying multifunctional GnRH systems in future.