We tested the hypothesis that changes in phosphorylation of the sarcoplasmic reticulum (SR) protein, phospholamban (PlB) and myofibrillar proteins troponin I (TnI) and C protein are responsible for the decreased relaxant response to isoproterenol in cardiac hypertrophy and failure induced by ascending aortic banding in rats. In isolated perfused heart preparations under maximal isoproterenol stimulation, the capacity for in vitro cAMP-dependent phosphorylation of PlB was significantly increased at the compensatory stage of hypertrophy (126–130%, P<0.001), but decreased with failure (70–76%, P<0.001). Phosphorylation of TnI also decreased in the failing hearts, however to a lesser extent (80–83%, P<0.05). No significant hypertrophy-related difference was evident in isoproterenol-induced phosphorylation of C protein. The relative tissue level of PlB was increased (150–168%, P<0.001) in hypertrophied and decreased (71–83.8%, P<0.05) in failing hearts compared with the respective age-matched sham-operated controls (100%). As a percentage above baseline, the maximal isoproterenol-induced increase in the EC50 of the SR Ca2+ pump in response to phosphorylation of PlB was 38.5±1.1% for sham-operated rats, and 26.0±3.8% and 15.4±4.2% for hypertrophied and failing hearts respectively. As a consequence, linear correlation was observed between the maximal increase in EC50 and the maximal rate of relaxation [(–dP/dt)/DevP] upon isoproterenol stimulation, declining with progressive hypertrophy to failure. These data suggest that hypertrophy-induced alterations in PlB phosphorylation and protein level contribute to the diminished relaxant response of the hypertrophied and failing heart to adrenergic agonists.