The effects of b-adrenergic stimulation on phospholamban phosphorylation and Ca2+-ATPase activity were studied in intact myocardium. Isolated guinea pig ventricles were perfused via the coronary arteries with Pi after which membrane vesicles, comprised primarily of sarcoplasmic reticulum, were isolated by differential centrifugation. Isoproterenol perfusion icreased 32P incorporation into membrane proteins of apparent iM,= 22,000 and 8,000, which have been shown to be the predominant proteins phosphorylated in sarcoplasmic reticulum by CAMP-dependent protein ki-nase." P incorporation was increased%% fold and 7.1-fold after 60-s exposure to 10 and 100 nM isoproterenol, respectively. In the same membrane vesicles, Ca'+-ATPase activity was increased by 30 and 52%, respectively. The increases in phospholamban phosphorylation and Caz+-ATPase activity correlated in time and concentration dependence with increases in the rate of myocardial relaxation. These biochemical changes did not correlate well with positive inotropy. Similar effects were produced by histamine, 1 PM. The onset of the effects of isoproterenol on phospholamban phosphorylation and Ca"-ATPase activity closely paralleled the relaxation effects. However, reversal of the relaxant effects occurred more rapidly than the return of 32P incorporation and CaZ+-ATPase activity to control levels. These results provide evidence that P-adrenergic stimulation results in phospholamban phosphorylation and increased Ca2'-ATPase activity in intact, functioning ventricular myocardium. These findings support he hypothesis that stimulation of sarcoplasmic reticulum Ca2+ transport, mediated by CAMP-dependent phosphorylation of phospholamban, may underlie the relaxant effects of &adrenergic stimulation in mammalian myocardium.