Homologous desensitization of ovarian LH/hCG-responsive adenylyl cyclase in cell-free systems has been reported to be dependent upon either ATP or GTP. We investigated LH/hCG-dependent desensitization of adenylyl cyclase in rabbit or pig ovarian follicular membranes and included adenyl-5'-yl imidodiphosphate to prevent nucleotide triphosphate degradation. It was found that GTP supported LH/hCG-induced desensitization with an apparent Km of approximately 0.1 microM in rabbit or pig ovarian membranes. Other nucleotide triphosphates were 100-1000 times less potent than GTP in supporting desensitization. Several nonhydrolyzable GTP analogs and the GDP analog guanosine-5'-O-(2-thiodiphosphate) would not support hCG-induced desensitization of ovarian adenylyl cyclase. Instead, these guanine nucleotide analogs were all inhibitors of GTP-supported hormone-dependent desensitization. Cholera toxin had no effect on LH-dependent desensitization. These results establish that GTP is the preferred nucleotide for homologous desensitization of the LH/hCG-sensitive adenylyl cyclase and that the GTP-dependent mechanism differs from that typically associated with guanine nucleotide-binding proteins. The GTP-dependent mechanism of desensitization of the LH receptor distinguishes it from the ATP-dependent desensitization of the beta-adrenergic receptor.