These studies examined the structural specificity for guanine nucleotide-facilitated hormonal activation and guanine nucleotide stabilization of cardiac adenylate cyclase. 1. The phosphonate analogues of GTP, p[CH(2)]ppG (guanosine 5'-[betagamma-methylene]-triphosphate) and pp[CH(2)]pG (guanosine 5'-[alphabeta-methylene]triphosphate), were the most effective activators of adenylate cyclase. Other nucleotides producing significant activation (P<0.01) were, in decreasing order of activation: ITP, GDP, GMP, GTP, XTP, CTP, p[NH]ppG (guanosine 5'-[betagamma-imido]triphosphate), dGTP and 2'-O-methyl-GTP. Guanosine, cyclic GMP, UTP and ppppG (guanosine tetraphosphate) had no effect, and 7-methyl-GTP caused a decrease in the activity. 2. Preincubation of membranes at 37 degrees C for 15min before assay at 24 degrees C produced an 80% decrease in adenylate cyclase activity, and preincubation with p[CH(2)]ppG and pp[CH(2)]pG protected and resulted in a net increase in activity. Other nucleotides that completely or partially preserved activity in decreasing order of effectiveness were p[NH]ppG, GDP, GTP, dGTP, ITP, ppppG, 2'-O-methyl-GTP, GMP, CTP and XTP. Several compounds had no effect, including guanosine, cyclic GMP and UTP, whereas preincubation with 7-methyl-GTP produced a further decrease (P<0.05) in activity. 3. The concentration-dependence for activation and stabilization by the naturally occurring guanine nucleotides was examined in the absence of a regenerating system and revealed GMP to have no stabilizing effect and to be less potent than either GDP or GTP in activating adenylate cyclase. 4. A significant correlation (r=0.90) was found between the properties of activation and stabilization for the compounds examined. These findings are consistent with there being a single nucleotide site through which both the activation and stabilization of adenylate cyclase are mediated.