The two native forms of GnRH, salmon GnRH and chicken GnRH-II, in the brain and pituitary of goldfish are both active in stimulating gonadotropin-II (GTH-II) and GH release. The objective of the present study was to characterize GnRH antagonists for their ability to inhibit sGnRH- and cGnRH-II-induced GTH-II and GH release in goldfish using a pituitary fragments perifusion system. Contrary to expectations, putative GnRH antagonists with D-Arg6 stimulated GTH-II and GH release in nearly all cases. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mammalian (m) GnRH inhibited sGnRH- and cGnRH-II-stimulated GTH-II release in a dose-dependent manner, with ED50 values of 242 +/- 48 and 169 +/- 17 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH also inhibited GH release stimulated by sGnRH (ED50, 128 +/- 74 nM) and cGnRH-II (ED50, 157 +/- 67 nM). The degree of inhibition was higher in sexually regressed fish compared to postspawning fish. [D-p-Glu1,D-Phe2,D-Trp3,6]mGnRH suppressed both sGnRH- and cGnRH-II-induced GTH-II release with ED50 values of 326 +/- 96 and 249 +/- 74 nM, respectively. [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]sGnRH inhibited sGnRH and cGnRH-II stimulated GTH-II release, but stimulated GH release. On the other hand, [Ac-D(2)-Nal1,4Cl-D-Phe2,D-(3)Pal3,6,Arg5,D-A la10]mGnRH weakly stimulated GTH-II release, but strongly inhibited basal GH release. These results indicate that [Ac-delta 3-Pro1,4FD-Phe2,D-Trp3,6]mGnRH has clear antagonistic activity on sGnRH and cGnRH-II stimulation of GTH-II and GH release in vitro. The differential actions of a few GnRH analogs on GTH-II and GH release indicate that the properties of the GnRH receptors on GTH and GH cells may be different. The amino acid in position 6 plays an important role in determining the nature of intrinsic activity of GnRH peptides, and substitution of D-Arg6 normally produces agonistic analogs.