To differentiate the effects of GDP and GTP on adenylyl cyclase regulation, adenylyl cyclase in canine sarcolemmal membranes was studied under conditions where only 3-12% of added GDP was converted to GTP by membrane-associated nucleoside diphosphate kinase. Adenylyl cyclase was stimulated up to 180% by GDP at 7-fold lower concentrations than required for stimulation by GTP (GDP half-maximal activation, 120 nM; GTP half-maximal activation, 830 nM). Transphosphorylation of GDP to GTP was blocked completely by the addition of 3 mM UDP. However, UDP did not affect GDP-mediated adenylyl cyclase activation, and guanosine 5'-O-(2-thiodiphosphate) had the same effect on adenylyl cyclase activity as did GDP, indicating that GDP-mediated stimulation of adenylyl cyclase was not due to transphosphorylation of GDP to GTP. Carbachol inhibited GDP-stimulated adenylyl cyclase activity even without addition of GTP; however, this inhibition was clearly dependent upon the endogenous formation of GTP. Half-maximal adenylyl cyclase inhibition by carbachol required the addition of either 330 nM GDP or 25 nM GTP. Taking into account a 3-12% conversion of GDP to GTP by membrane-associated nucleoside diphosphate kinase, sufficient GTP was generated from GDP to support receptor-mediated inhibition of adenylyl cyclase. In addition carbachol-mediated adenylyl cyclase inhibition in the presence of GDP, but not GTP, was blocked completely by 3 mM UDP. In conclusion, GDP-activated adenylyl cyclase could be inhibited by carbachol in the presence of GTP concentrations that were 34-fold below the concentrations needed for GTP-mediated activation of stimulatory guanine nucleotide-binding proteins. In addition, at low GTP concentrations carbachol reduced adenylyl cyclase to levels below "basal" activity (activity in the absence of guanine nucleotides). Although indirectly, these results suggest that carbachol-mediated inhibition of adenylyl cyclase may be independent of Gs activity and possibly due to direct interaction of inhibitory guanine nucleotide-binding proteins and adenylyl cyclase.