Gonadotropin-releasing hormone (GnRH) controls reproductive functions in vertebrates. In the present study, two distinct homologues (Ci-GnRHR1 and Ci-GnRHR2) of the vertebrate GnRH receptor (GnRHR) were identified by cDNA cloning from the neural complex of the ascidian Ciona intestinalis. The Ci-GnRHR1 and Ci-GnRHR2 genes are closely linked to each other and form a cluster with another GnRHR-like gene in the genome. Ci-GnRHR1 and Ci-GnRHR2 are more closely related to vertebrate GnRHRs than to the GnRHR-related protein of Drosophila. The phylogenetic analysis indicates that vertebrates and urochordates independently acquired multiple GnRHRs by gene duplications that occurred during the evolution of each lineage. A voltage clamp of Xenopus oocytes injected with synthetic Ci-GnRHR1 mRNA revealed inward currents in response to an ascidian form of GnRH, suggesting that Ci-GnRHR1 is a bona-fide GnRHR. Expression patterns of Ci-GnRHR1 and Ci-GnRHR2 suggest that a GnRH signaling system is involved in regulation of neuronal and reproductive processes as well as in other physiological functions in ascidians.