Phosphorylation/dephosphorylation of Ca²⁺ 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 Ca²⁺ handling in normal and diseased hearts remains poorly understood. We have investigated the effects of protein phosphatases PP1 and PP2A on spontaneous Ca²⁺ sparks and SR Ca²⁺ load in myocytes permeabilized with saponin. Exposure of myocytes to PP1 or PP2A caused a dramatic increase in frequency of Ca²⁺ sparks followed by a nearly complete disappearance of events. These effects were accompanied by depletion of the SR Ca²⁺ stores, as determined by application of caffeine. These changes in Ca²⁺ release and SR Ca²⁺ 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 Ca²⁺ release leading to depleted SR Ca²⁺ stores in cardiac myocytes.