Gonadotropin-releasing hormone (GnRH) plays pivotal roles in the regulation of vertebrate reproduction through binding to its specific membrane receptor. Within the past few years, substantial evidence has accumulated that more than one GnRH receptor (GnRH-R) is expressed in individual vertebrate species. Two GnRH-Rs, termed GnRH-R1 and GnRH-R2, have been identified in a teleost, the medaka Oryzias latipes. Here we describe the identification and characterization of a novel third member of GnRH-R, designated GnRH-R3, in the medaka. GnRH-R3 share high sequence homology (77% amino acid identity in the transmembrane domain) with GnRH-R1. Phylogenetic analysis and genetic mapping demonstrated that both GnRH-R1 and GnRH-R3 were orthologous to the type 2 GnRH-R in primates and that these two medaka receptors were duplicates resulting from the genome-wide duplication within the teleost lineage. GnRH-R3, however, contained three introns, whereas GnRH-R1 had only two. Moreover, unlike GnRH-R1, GnRH-R3 exhibited an approximately equal selectivity for two of three native GnRH forms in the medaka, chicken-II-type GnRH (cGnRH-II) and salmon-type GnRH (sGnRH), and a less sensitivity for the other form, medaka-type GnRH. GnRH-R3 was found to be expressed throughout the brain, and thus appeared to mediate the neuromodulatory functions of both cGnRH-II and sGnRH. These data identify GnRH-R3 as a new member of GnRH-R that arose in a recent genome duplication but has distinctive genomic structure and functional characteristic.