Guanosine 5'-tetraphosphate (GTP4) stimulated mammalian adenylate cyclase activity at concentrations down to 1 micronM. Greater stimulatory activity was apparent with lung than with heart, brain or liver from the rat. At a concentration of 0.1 mM, GTP4 stimulated lung adenylate cyclase activity from rat, guinea pig and mouse about four-fold. Other guanine nucleotides such as GTP, GDP, GMP, guanosine 3',5'-monophosphate and 5'-guanylylimidodiphosphate (GMP.PNP) also stimulated mammalian adenylate cyclase activity. EMP.PNP irreversibly activated, whereas GTP4 and GTP reversibly activated adenylate cyclase. Adenosine 5'-tetraphosphate (ATP4) stimulated rat lung and liver but inhibited rat heart and brain adenylate cyclase activities. Lung from guinea pig and mouse were not affected by ATP4. The formation of cyclic AMP by GTP4-stimulated rat lung adenylate cyclase was verified by Dowex-50 (H+), Dowex 1-formate and polyethyleneimine cellulose column chromatography. GTP4 was at least three times more potent than 1-isoproterenol in stimulating rat lung adenylate cyclase activity. The beta-adrenergic receptor antagonist propranolol blocked the effect of 1-isoproterenol but not that of GTP4, thus, suggesting that GTP4 and beta-adrenergic agonists interact with different receptor sites on membrane-bound adenylate cyclase. Stimulation of rat lung and liver adenylate cyclase activities with 1-isoproterenol was potentiated by either GTP4 or GMP.PNP, thus indicating that GTP4 resembles other guanine nucleotides in their capacity to increase the sensitivity of adenylate cyclase to beta-adrenergic agonists. Stimulation of adenylate cyclase activity by guanine derivatives requires one or more free phosphate moieties on the 5 position of ribose, as no effect was elicited with guanine, guanosine, guanosine 2'-monophosphate, guanosine 3'-monophosphate or guanosine 2',5'-monophosphate. Ribose, ribose 5-phosphate, phosphate and pyrophosphate were inactive. Pyrimidine nucleoside mono-, di-, tri- and tetraphosphates elicited negligible effects on mammalian adenylate cyclase activity.