In G protein-coupled receptors, neurotransmitter-induced binding of GTP to G proteins triggers the activation of effector systems while simultaneously decreasing the affinity of the transmitter for its specific binding site within the receptor-G protein complex. In the present study we show that, in the chick optic tectum, guanine nucleotides inhibit the binding of the glutamate analog, kainate, and activate adenylate cyclase by different mechanisms and acting on different sites. GMP-PNP, a non-hydrolyzable analog of GTP, binds tightly to G proteins so that the binding is stable even after exhaustive washing. By use of this property, we have prepared membrane samples in which G protein GTP-binding sites are pre-saturated with GMP-PNP. Experiments carried out with these membranes show that GMP-PNP, GDP-S and GMP inhibit the binding of [3H]kainate by interacting with site(s) unrelated to G proteins, whereas GMP-PNP activates adenylate cyclase activity by binding to G proteins.