Binding of the GnRH agonist [DAla6,NMe-Leu7,Pro9Net]GnRH to bovine anterior pituitary membranes is inhibited by guanyl nucleotides. The effect of guanyl nucleotides is temperature dependent, in that significant binding inhibition is observed when the receptor-hormone interaction is measured at 37 C, and no inhibition is seen at 4 C. Micromolar concentrations of the nonhydrolyzable GTP analog 5'-guanylylimidodiphosphate [Gpp(NH)p] displace the bound agonist in a dose-dependent manner, with half-maximal displacement occurring in a concentration range of 0.1-0.5 microM, and maximum displacement occurring at a concentration of 50 microM Gpp(NH)p. At a concentration of 50 microM, the other nucleotides GTP and GDP inhibit binding to a lesser extent, while GMP, cGMP, 5'-adenylylimidodiphosphate [App(NH)p], ATP, and cAMP have no effect on the binding. At 37 C, Gpp(NH)p reduces the affinity of the agonist by a factor of 6 and increases its dissociation rate. In the presence of Gpp(NH)p at 37 C, there is also a 2-fold increase in the total number of binding sites. Under the same conditions as those used for the agonist, there is no displacement of the bound antagonist [Ac-D2Nal1,4ClDPhe2,D3Pal3,DLys6,Lys8,D Ala10]-GnRH by doses up to 50 microM Gpp(NH)p. The modulation of the binding of the agonist, but not that of the antagonist, by guanyl nucleotides is characteristic of receptors that are coupled to GTP-binding proteins. Thus, the GnRH receptor appears to be coupled to a GTP-binding protein that may play a role in the mechanism of action of GnRH at the pituitary.