Binding of (3H)GTP to solubilized preparations of myocardial adenylate cyclase, partially purified by DEAE-cellulose chromatography, as been studied in an attempt to gain further insight into the mechanisms by which guanine nucleotides regulate adenylate cyclase activity. Although several peaks of (3H)GTP-binding activity were present in crude preparations of solubilized myocardium, one peak was associated with the adenylate cyclase peak. Binding of (3H)GTP to this material was rapid (equilibrium within 3 min at 37 degrees) and reversible and not associated with nucleotide hydrolysis. Scatchard analysis revealed a single class of (3H)GTP binding sites with KA = 3 x 10-6 M-1 and total binding capacity of 50 pmol per mg of protein. The GTP analog Gpp(NH)p competed for the sites with an affinity somewhat lower than GTP, although its ability to activate the adenylate cyclase was far greater. GTP and other guanine nucleotides activated the soluble cyclase only weakly, although they antagonized competitively enzyme stimulation by Gpp(NH)p. Ability of GTP and other nucleotides to compete with (3H)GTP for binding sites and to antagonize competitively adenylate cyclase activation by Gpp(NH)p were directly parallel. The potency series was GTP = GDP = dGTP greater than GMP greater than ITP greater than UTP, CTP. Dissociation constants of nucleotides for the sites determined by inhibition of (3H)GTP binding and inhibition of Gpp(NH)p activation of cyclase agreed closely. Gpp(NH)p dose-response curves for activation of adenylate cyclase and inhibition of (3H)GTP binding were superimposable.