Recent studies have demonstrated that the G protein-coupled receptor 54 (GPR54) and its natural ligand kisspeptin (KiSS) play important roles in the control of puberty and reproductive maturation in mammals. In addition, GPR54 mRNA has been localized on the neurons expressing three GnRH mRNAs in tilapia (Parhar et al., 2004). However, localization of GPR54 protein on GnRH neuronal elements has not been reported. The present study was designed to localize GPR54 protein expression on GnRH neurons in the Atlantic croaker, Micropogonias undulatus. GPR54 protein was co-localized with seabream GnRH in the preoptic-anterior hypothalamic area (POAH), with chicken GnRH-II (cGnRH-II) in midbrain tegmentum and with salmon GnRH (sGnRH) in terminal nerve ganglion cells present in the posterior olfactory bulb. GPR54 expression was quantified by Western blot analysis and showed significantly higher expression in the POAH of females undergoing early puberty compared to the immature individuals. This activation of GPR54 signaling during early puberty may be an important component of the KiSS-GPR54-GnRH neuroendocrine pathway in fish similar to the situation in mammals. This possibility is further supported by the recent cloning of a KiSS-like cDNA in Atlantic croaker. Moreover, co-localization of GPR54 protein on cGnRH-II and sGnRH suggests that the KiSS-GPR54 system may regulate multiple GnRHs in fish and possibly other vertebrate groups.