Synthetic ligands have been identified that reset and amplify the cycle of pulsatile GH secretion by interacting with the orphan GH-secretagogue receptor (GHS-R). The GHS-R is rhodopsin like, but does not obviously belong to any of the established G protein-coupled receptor (GPCR) subfamilies. We recently characterized the closely related orphan family member, GPR38, as the motilin receptor. A common property of both receptors is that they amplify and sustain pulsatile biological responses in the continued presence of their respective ligands. To efficiently identify additional members of this new GPCR family, we explored a vertebrate species having a compact genome, that was evolutionary distant from human, but where functionally important genes were likely to be conserved. Accordingly, three distinct full-length clones, encoding proteins of significant identity to the human GHS-R, were isolated from the Pufferfish (Spheroides nephelus). Southern analyses showed that the three cloned Pufferfish genes are highly conserved across species. The gene with closest identity (58%) was activated by three synthetic ligands that were chosen for their very high selectivity on the GHS-R as illustrated by their specificity in activating the wild-type human GHS-R but not the E124Q mutant. These results indicate that the ligand activation domain of the GHS-R has been evolutionary conserved from Pufferfish to human (400 million years), supporting the notion that the GHS-R and its natural ligand play a fundamentally important role in biology. Furthermore, they illustrate the power of exploiting the compact Pufferfish genome for simplifying the isolation of endocrinologically important receptor families.