Homologous desensitization of the ovarian LH-sensitive adenylyl cyclase in cell-free systems is known to be dependent on micromolar concentrations of GTP. In this study, we sought to determine whether the nucleotide requirement of GTP for desensitization is complete and specific. LH-dependent desensitization of the adenylyl cyclase of pig ovarian follicular membranes was examined without adding adenyl-5'-yl imidodiphosphate [AMP-P(NH)P] to prevent the degradation of nucleotide triphosphates. GTP at 0.1 mM or higher [in the absence of AMP-P(NH)P] was able to support the same amount of desensitization (30-35%) that occurs with 1 mM AMP-P(NH)P and micromolar concentrations of GTP. ATP, UTP, and CTP also supported maximal desensitization, but were 5-30 times less potent than GTP. The ED50 value for GTP (14 microM) was about 100 times higher than when the reaction was performed in the presence of 1 mM AMP-P(NH)P; the ED50 values for UTP (70 microM) and CTP and ATP (400 microM) were only 2-5 times higher than when AMP-P(NH)P was included. Incubation of [alpha-32P]GTP with pig follicular membranes demonstrated that 1 mM GTP was stable, whereas 10 microM or less was rapidly degraded unless 1 mM AMP-P(NH)P was included. The specificity of GTP for supporting desensitization was also demonstrated in experiments where guanyl-5'-yl imidodiphosphate, the nonhydrolyzable GTP analog, inhibited LH-induced desensitization which otherwise would have occurred in the presence of ATP, CTP, or UTP. These studies establish the complete and specific requirement of GTP for supporting desensitization of ovarian LH-responsive adenylyl cyclase.