We previously reported that a blockade of GnRH receptor activation inhibited the already-initiated C-kinase pathway(s). We tried to investigate whether this finding is a general phenomenon or not. In this study, we employed three GnRH antagonists, [D-Phe2,Pro3,D-Phe6]-GnRH, [Ac-D-Nal-Ala1,D-pCl-Phe2,D-Ser(Rha)6]-GnRH, and [Ac-D-p-Cl-Phe1,2,D-Trp3,D-Lys6,D-Ala10]-GnRH (referred to as #1-, #2-, #3-GnRH antag., respectively). Each antagonist was examined for its potency against GnRH by analyzing its inhibitory effect on LH release from pituitary gonadotrophs as well as on the increase in the cytosolic Ca2+ concentration. As a result, the #1-GnRH antag. was found to be weaker than the other two compounds. Consistent with a previous study, the #3-GnRH antag. inhibited the action of TPA on LH release. However, independently of their potency as GnRH-antagonists, the two other antagonists had no inhibitory effect on TPA-induced LH release. While it is generally accepted that the C kinase pathway plays a major role in the GnRH-induced LH release, not all GnRH antagonists can inhibit LH release by blocking the already-activated C kinase system.