It is generally believed that hypothalamic GHRH activates GHRH receptor (GHRHR) to stimulate GH synthesis and release in the pituitary of mammals. However, the identity of the endogenous ligand of GHRHR is still unresolved in submammalian vertebrates including birds. In this study, we have successfully identified the chicken GHRH (cGHRH) gene, which consists of seven exons including two exons (exons 4 and 5) coding for the predicted mature GHRH peptide of 47 amino acids. Interestingly, the differential usage of splice donor sites at exon 6 results in the generation of two prepro-GHRHs (172 and 188 amino acids in length) with different C-terminal tails. Similar to mammals, cGHRH was detected to be predominantly expressed in the hypothalamus by RT-PCR assay. Using the pGL3-CRE-luciferase reporter system, we further demonstrated that both the synthetic cGHRH peptides (cGHRH(1-47) and cGHRH(1-31)) and conditioned medium from CHO cells expressing cGHRH could strongly induce luciferase activity via activation of cGHRHR, indicating that cGHRH could bind cGHRHR and activate downstream cAMP-protein kinase A signaling pathway. Using the same system, cGHRH-like peptide was also shown to be capable of activating cGHRHR in vitro. As in chicken, a conserved GHRH gene was identified in the genomes of lower vertebrate species including zebrafish, fugu, tetraodon, and Xenopus by synteny analysis. Collectively, our data suggest that GHRH, perhaps together with GHRH-like peptide (chicken/carp-like), may function as the authentic endogenous ligands of GHRHR in chicken as well as in other lower vertebrate species.