The sarcoplasmic reticulum (SR) plays a critical role in mediating cardiac contractility and its function is abnormal in the diabetic heart. However, the mechanisms underlying SR dysfunction in the diabetic heart are not clear. Because protein phosphorylation regulates SR function, this study examined the phosphorylation state of phospholamban, a key SR protein that regulates SR calcium (Ca²⁺) uptake in the heart. Diabetes was induced in male Sprague-Dawley rats by an injection of streptozotocin (STZ; 65 mg kg^–1 i.v.), and the animals were humanely killed after 6 weeks and cardiac SR function was examined. Depressed cardiac performance was associated with reduced SR Ca²⁺-uptake activity in diabetic animals. The reduction in SR Ca²⁺-uptake was consistent with a significant decrease in the level of SR Ca²⁺-pump ATPase (SERCA2a) protein. The level of phospholamban (PLB) protein was also decreased, however, the ratio of PLB to SERCA2a was increased in the diabetic heart. Depressed SR Ca²⁺-uptake was also due to a reduction in the phosphorylation of PLB by the Ca²⁺-calmodulin-dependent protein kinase (CaMK) and cAMP-dependent protein kinase (PKA). Although the activities of the SR-associated Ca²⁺-calmodulin-dependent protein kinase (CaMK), cAMP-dependent protein kinase (PKA) were increased in the diabetic heart, depressed phosphorylation of PLB could partly be attributed to an increase in the SR-associated protein phosphatase activities. These results suggest that there is increased inhibition of SERCA2a by PLB and this appears to be a major defect underlying SR dysfunction in the diabetic heart. (Mol Cell Biochem 261: 245–249, 2004)