Adenosine inhibits membrane adenylate cyclase from rabbit heart and considerably alters its regulatory properties. In the presence of 1 mM adenosine the enzyme activation by isoproterenol and guanyl-5'-ylimidodiphosphate is sharply decreased, while that by fluoride is practically completely eliminated. The values of the apparent rate constants for the enzyme inhibition by adenosine with respect to the basal activity and the activity in the presence of NaF are equal to 1.6 . 10(-4) and 5.9 . 10(-5) M, respectively. The inhibiting effect of adenosine is rapid and irreversible and is not changed in the presence of 2 mM theophylline and during adenylate cyclase solubilization. It is assumed that the enzyme inhibition is mediated by the P-sites. An increase in ATP concentration causes an increase in the enzyme activation by fluoride. At low ATP concentrations (10(-5) - 2 . 10(-5) M) adenosine inhibits the activation by fluoride practically completely, while at increasing ATP concentrations the inhibition of the fluoride-stimulated activity of adenylate cyclase is decreased. The effect of adenosine is not competitive with respect to ATP. The increased activation of adenylate cyclase by fluoride in the presence of ATP can be due to the fact that the enzyme affinity for the substrate is decreased in the presence of NaF. Adenosine, which acts presumably at the allosteric site, causes an additional decrease of the fluoride-stimulated adenylate cyclase affinity for ATP. It is also assumed that within the adenylate cyclase complex there probably exists a regulatory site, whose binding to ATP increases the activation of adenylate cyclase by fluoride; the adenosine binding to the P-sites inhibits this activation.