In most experiments done in cell-free systems, the LH/CG-induced desensitization of the ovarian LH/CG-responsive adenylyl cyclase has been reported to be dependent on GTP. Little is known, however, about the molecular basis of this phenomenon or about the FSH-induced desensitization of the FSH-responsive adenylyl cyclase. We report here that, contrary to most previous findings, ATP is required for desensitization of the LH/CG- and FSH-responsive adenylyl cyclase in human kidney cells stably transfected with the complementary DNAs for the rat LH/CG or FSH receptor. This requirement does not seem to be peculiar to transfected cells because under our experimental conditions ATP is also preferred over GTP for the human CG-induced desensitization of the LH/CG-responsive adenylyl cyclase in highly purified plasma membranes from MA-10 Leydig tumor cells. Maximal desensitization of both FSH- and LH/CG-sensitive adenylyl cyclase in membranes from the transfected cells was achieved with millimollar concentrations of Mg2+ and ATP and did not appear to correlate with activation of the enzyme. In both of these systems, GTP, uridine triphosphate, and cytidine triphosphate were not able to substitute for ATP. In MA-10 membranes, however, there was some desensitization even without added nucleotide triphosphates, and ATP was more potent than GTP. Last, desensitization of the gonadotropin-sensitive adenylyl cyclase could not be explained by a decrease in the functional activities of stimulatory guanine nucleotide binding protein or of the catalytic moiety of the enzyme. A change in the functional properties of the gonadotropin receptors appears to be the most likely mechanism for desensitization.