This study was aimed to elucidate whether GDP can mediate hormonal signal to adenylate cyclase in hepatic glucagon sensitive adenylate cyclase with ATP as substrate. Conversion of added GDP to GTP catalyzed by nucleoside diphosphate kinase was suppressed to less than 0.3% of added GDP by including UDP. Inhibition of this enzyme activity by UDP was accompanied by a preferential loss of the stimulatory effect of glucagon plus GDP on cyclase activity without changes in effects of glucagon plus GTP, glucagon plus guanosine 5'-(beta, gamma-imino)triphosphate, and NaF. Under this condition, i.e. in the presence of UDP, GDP competitively inhibited the actions of GTP (Ki for GDP, 1 microM) and guanosine 5'-(beta, gamma-imino)triphosphate in the presence of glucagon, the inhibition being complete at high GDP concentrations. GDP also inhibited cyclase activity stimulated by NaF with UDP but did only slightly without UDP. It was demonstrated that nucleoside diphosphate kinase is located in membranes in addition to cytosol fraction. However, the activity of membrane-associated enzyme was not affected by the addition of glucagon. Based on these observations, it is concluded that GDP is unable to mediate hormonal signal to adenylate cyclase and that it acts as an inhibitor of cyclase activity stimulated by GTP or its analog along with hormone. The results suggest a possible role of membrane-associated nucleoside diphosphate kinase in determining GTP and GDP levels at or near their binding site so as to replenish GTP and, thereby, decrease the inhibitory action of GDP when hormone is present.