The binding and activity of gonadotropin-releasing hormone (GnRH) were characterized in the mature gilthead seabream (Sparus aurata) ovary by use of an analogue of salmon GnRH([D-Arg6,Trp7,Leu8,Pro9-N(Et)]GnRH, sGnRH-A) as labeled ligand. The binding of 125I-sGnRH-A to the seabream ovarian membrane preparation was saturable, displaceable, reversible, and dependent on time, temperature and tissue concentration. Addition of unlabeled s-GnRH-A displaced the radio-ligand in a dose-related manner, indicating the presence of one class of high-affinity binding sites with an equilibrium dissociation constant of 45.5 +/- 6.2 nM. Addition of other GnRH peptides, including salmon GnRH ([Trp7,Leu8]GnRH) and chicken GnRH-II ([His5,Trp7,Tyr8]GnRH), also displaced 125I-sGnRH-A; all these peptides bound with lower affinities than sGnRH-A to the seabream ovarian binding site. In this study, we also demonstrated the presence of compounds with GnRH-like activity in the ovary of seabream. Seabream ovarian extract stimulated pituitary gonadotropin release from the goldfish pituitary and displaced 125I-sGnRH-A binding in the seabream ovary. Furthermore, addition of sGnRH-A to cultured seabream oocytes directly stimulated reinitiation of oocyte meiosis, as indicated by germinal vesicle breakdown. Overall, the present study characterizes GnRH-binding sites in the seabream ovary and supports the hypothesis that GnRH or compounds with GnRH-like activity play an autocrine/paracrine role in the regulation of ovarian function in the seabream ovary.