The relationship between changes in the myocardial concentration of adenosine 3':5'-cyclic monophosphate (cyclic AMP) and cardiac contractility was studied in guinea pig and rat myocardium. When isolated perfused guinea pig heart were perfused with 10-5-M papaverine, a potent inhibitor of cyclic AMP phosphodiesterase activity, myocardial cyclic AMP concentration increased significantly from 1.7 plus and minus 0.2 (SE) pmoles/mg protein (N equal 12) to 3.3 plus and minus 0.2 pmoles/mg protein (N equal 12), and the percent of phosphorylase aual 6) (P less than 0.01). However, perfusion with papaverine had no effect on contractility in the absence or the presence of exogenous epinephrine. In perfused rat hearts, 10-5 M glucagon increased myocardial cyclic AMP concentration from 1.5 plus and minus 0.1 pmoles/mg protein (N equal 12) to 2.6 plus and minus 0.1 pmoles/mg protein (N equal 12) (P less than 0.001). In contrast, cyclic AMP levels did not increase detectably in guinea pig heart perfused with glucagon. Glucagon increased adenylate cyclase activity more than twofold in rat myocardial broken cell preparations but failed to stimulate the enzyme in preparations from guinea pigs. Despite these differences, the positive inotropic effects of glucagon on rat and guinea pig hearts were very similar over a wide dose range. Thus, with both papaverine and glucagon, changes in cardiac contractility were dissociated from stimulation of adenylate cyclase activity, increases in myocardial cyclic AMP levels, and conversion of phosphorylase b to phosphorylase a in perfused hearts.