Phosphorylation/dephosphorylation of Ca transport proteins by cellular kinases and phosphatases plays an important role in regulation of cardiac excitation-contraction coupling; furthermore abnormal protein kinase and phosphatase activities have been implicated in heart failure. However, the precise mechanisms of action of these enzymes on intracellular Ca handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca sparks and SR Ca load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca stores, as determined by application of caffeine. These changes in Ca release and SR Ca load could be prevented by the inhibitors of PP1 and PP2A phosphatase activities okadaic acid and calyculin A. At the single channel level, PP1 increased the open probability of RyRs incorporated into lipid bilayers. PP1-medited RyR dephosphorylation in our permeabilized myocytes preparations was confirmed biochemically by quantitative immunoblotting using a phosphospecific anti-RyR antibody. Our results suggest that increased intracellular phosphatase activity stimulates RyR-mediated SR Ca release leading to depleted SR Ca stores in cardiac myocytes.