In vertebrates, gonadotropin-releasing hormone (GnRH), which is synthesized in the brain, is a key peptide involved in gonadal maturation regulated by the brain-pituitary-gonadal axis. GnRH isoforms and their primary structures have recently been determined in two species of non-chordates, the octopus (Octopus vulgaris) and sea hare (Aplysia californica), which are mollusks. Octopus and sea hare GnRHs are dodecapeptides that contain the structural core of chordate GnRH; however, chordate GnRHs, including tunicate GnRH, are decapeptides. In this study, we examined a GnRHlike peptide in the swordtip squid, Loligo edulis, to provide information on the structural evolution of GnRH in non-chordates. We isolated the full-length cDNA of a GnRH-like molecule from the central nervous system (CNS) of the squid. The open reading frame of this cDNA encodes a protein of 90 amino acids, which consists of a putative signal peptide, a GnRH dodecapeptide, a processing site, and a GnRH-associated peptide. This architecture is generally conserved in chordates. Compared to octopus GnRH, Squid GnRH is identical in the deduced amino acid sequence of the peptide, and 80.5% similar in base sequence. In a phylogenetic analysis, prepro-GnRHs of octopus, sea hare, and squid were segregated from all chordate prepro-GnRHs, in a group designated GnRH5. The squid prepro-GnRH mRNA was expressed mainly in the CNS. This study is the first report of GnRH cDNA cloning in squid and the third in non-chordates.