RFamide-related peptides (RFRPs) regulate the release of various pituitary hormones in vertebrates. It is completely unknown how the functions of RFRPs vary among animal classes and whether vertebrate RFRPs are orthologous to each other and belong to the same peptide family. This report concerns identification of avian RFRP (gonadotropin-inhibitory hormone, GnIH) from the chicken. Chromosome-wide synteny conservation demonstrated the orthologous relationships among vertebrate RFRPs. The consensus motif for RFRP was modified to Pro-Xaa-Arg-Phe-NH2. We also describe the first identification of two distinct types of receptors for non-mammalian RFRP (RFRPR and NPFFR) from the chicken. Amino acid comparison revealed substantial differences in both termini of receptors among classes of vertebrates. The 5'-flanking regions of chicken RFRPR and NPFFR suggested their expressions in the pituitary gland, and this was confirmed by the RT-PCR analysis. Localizations of both chicken RFRP and its receptors were distinct from those of mammals. These results indicated that avian RFRP, unlike the mammalian one, directly acts on the pituitary gland via receptors to regulate gonadotropin release. It was also suggested that functional differences, especially between avian and mammalian RFRPs, are substantially due to divergences in the structures and expression sites of their receptors.