Vasotocin (VT) has been shown to influence various aspects of social and sexual behaviors in a broad range of vertebrate species, but less is known about the mechanisms through which this peptide modulates behavior. Additionally, much less is known about roles of isotocin (IT) in regulation of behavior. Grass puffer, Takifugu niphobles, has unique spawning behavior; spawning occurs on beach only for several days around the spring tide and is conducted by a group of 10-60 individuals, of which one is female. As a first step toward investigating the roles of VT and IT in this species' spawning behavior, we determined the structures of the VT and IT genes from grass puffer using! the genome resources of the closely related tiger puffer and green puffer. We then used these sequences to develop real-time PCR assays and examined changes in expression of the VT and IT genes over the spawning season. The structures of VT and IT genes are well conserved among three puffer species. Particularly, the sequence similarities between grass and tiger puffers were very high not only in the coding region (85-99%), but also in the non-coding regions (92-98%) that include the 5'-upstream regions. The levels of expression of VT gene increased in the brain of pre-spawning females. The levels of VT mRNA in the spawning females tended to be higher than that in the spawning males. In contrast, the levels of IT mRNA did not show such variation. The present results suggest that VT gene expression augments in the brain of females during the spawning period. The unique spawning behavior of grass puffer provides a useful model for studying the molecular mechanism of sexual behavior utilizing the genome resources of tiger puffer. (c) 2007 Elsevier Inc. All rights reserved.