Although Ca2+-transport activities in the cardiac sarcoplasmic reticulum (SR) have been shown to be depressed in chronic diabetes, the status of its regulatory mechanisms is not fully understood. Since Ca2+-calmodulin and cAMP-dependent protein kinases (CAMK and PKA) are known to stimulate SR function, it is possible that these enzymes may be altered in the diabetic heart. For this purpose, rats were made diabetic by an injection with streptozotocin; vehicle injected animals served as control. Some of the 4 week diabetic animals were treated with insulin (3 U/day) for 2 weeks. Hearts were removed at 6 weeks after the induction of diabetes and the ventricular tissue was used for either SR preparation or other biochemical determination. The decreased level of glucose, increased level of insulin and depressed ventricular function in diabetic animals were prevented by insulin treatment. Both Ca 2+-uptake and Ca2+-release activities in SR preparations from diabetic hearts were decreased. The SR protein content as estimated by Western blot analysis for Ca2+-pump ATPase, Ca2+-release channels and phospholamban proteins were also decreased in the diabetic heart . Both CAMK- and PKA-mediated protein phosphorylations were increased in the diabetic SR. These changes in the diabetic heart were associated with increased SR CAMK, PKA and phosphatase activities. Although insulin treatment of diabetic animals provided partial recovery of SR function, it had no effect on changes in CAMK and PKA activities. These results suggest marked changes in the regulatory mechanisms for SR function in the diabetic hearts.