Two forms of gonadotropin-releasing hormone (GnRH) were isolated from the gonads of the tunicate, Ciona intestinalis. The primary structure of the purified peptides was determined by MS and chemical sequence analysis. Both GnRH forms have blocked NH(2) and COOH termini, and their primary structures are identical to mammalian (mGnRH) and chicken I (cGnRH-I) forms reported previously in vertebrates. A total of 1.2 mg of purified cGnRH-I and 0.98 mg of mGnRH was obtained from 100 g of Ciona gonads. The physiological effects of native GnRHs included the induction of synthesis and secretion of sex steroids from ciona gonads and the secretion of luteinizing hormone from rat pituitary. These results suggest that the primary structure and functional roles of mGnRH and cGnRH-I have been highly conserved throughout evolution of chordates.