Three forms of gonadotropin-releasing hormone (GnRH) are found in vertebrates; these differ in amino acid sequence, localization, distribution, and embryological origin. We used northern blot analysis, and in situ hybridization to detect GnRH transcripts in various tissues in the large ascidian Halocynthia roretzi. We cloned a cDNA encoding two novel GnRHs, termed tGnRH-10 and tGnRH-11, from H. roretzi, with deduced amino acid sequences of QHWSYGFSPG and QHWSYGFLPG, respectively. Both GnRHs are highly similar to those of teleosts and tetrapods. For example, the tGnRH-10 sequence is 90% identical to seabream GnRH1, and tGnRH-11 is 90% identical to salmon GnRH3. The primary structure of the deduced preprotein is similar to that of chordate GnRHs and consists of a signal peptide, two decapeptides, up- and downstream processing sequences (containing lysine and arginine), and a GnRH-associated peptide. The transcripts of the H. roretzi GnRH gene were expressed in all tissues examined. Comparison of the signal peptide of the lamprey GnRH-II precursor with those of three forms from representative vertebrates revealed homology to GnRH2 precursors. These novel ascidian GnRHs offer a new perspective on the origin of vertebrate GnRH subtypes. We hypothesize that gnathostome GnRH2 was derived only from lamprey GnRH-II and that ancestral gnathostome GnRH, which produces neurons that originate in peripheral organs, gave rise to vertebrate GnRH1 and GnRH3 through whole-genome duplication.