Two GnRH peptides have recently been identified in brain extracts of the African catfish, chicken-II GnRH ([His5,Trp7,Tyr8]GnRH, cGnRH-II) and catfish GnRH ([His5,Asn8]GnRH, cfGnRH). Using three experimental approaches, we investigated whether both peptides are involved in the regulation of pituitary gonadotropin secretion. First, the presence of cfGnRH and cGnRH-II in the pituitary was studied by biochemical and immunocytochemical techniques, as GnRH reaches the pituitary via axonal transport in teleost fish. Pituitary extracts contained cfGnRH- and cGnRH-II-immunoreactive material, showing the same HPLC retention times as the respective synthetic GnRH peptides; cfGnRH was present in 37-fold higher amounts than cGnRH-II. Using single and double labeling immunocytochemical techniques, both peptides were localized in the same peptidergic nerve fibers and often within the same secretory granules in the vicinity of the gonadotropes. Second, the two peptides were tested for their capacity to induce an increased secretion of the LH-like gonadotropin-II (GTH-II). In vivo studies showed that both GnRHs released GTH-II, but 100-fold higher cfGnRH than cGnRH-II doses were necessary to induce similar increases in circulating GTH-II levels. In vitro experiments using pituitary tissue fragments in a perifusion system also revealed a clearly higher GTH-II-releasing capacity of cGnRH-II compared to that of cfGnRH. Third, the peptides were tested for their ability to displace [125I]salmon GnRH analog ([D-Arg6,Trp7,Leu8,Pro9-NEt] GnRH, sGnRHa), a high affinity GnRH receptor ligand, from catfish pituitary membrane preparations. Chicken GnRH-II competed with [125I]sGnRHa for pituitary GnRH-binding sites, whereas cfGnRH did so only slightly. The present data show that cGnRH-II is the more potent GTH-II secretagogue, although a role for cfGnRH in the regulation of GTH-II secretion cannot be excluded. The high biological activity of cGnRH-II may be related to the regulation of GTH-II secretion surges, such as those associated with spawning, whereas cfGnRH may be involved in regulating moderate changes in GTH-II plasma levels. The peptides' potency differences appear to be related to their different binding affinities for the pituitary GnRH receptor.