To decipher the mechanism(s) underlying glucocorticoid action on cardiac contractile function, this study investigated the effects of adrenalectomy and dexamethasone treatment on the contents of sarcoplasmic reticulum (SR) Ca²⁺-cycling proteins, their phosphorylation by endogenous Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II), and SR Ca²⁺ sequestration in the rat myocardium. Cardiac SR vesicles from adrenalectomized rats displayed significantly diminished rates of ATP-energized Ca²⁺ uptake in vitro compared with cardiac SR vesicles from control rats; in vivo administration of dexamethasone to adrenalectomized rats prevented the decline in SR function. Western immunoblotting analysis showed that the relative protein amounts of ryanodine receptor/Ca²⁺-release channel, Ca²⁺-ATPase, calsequestrin, and phospholamban were neither diminished significantly by adrenalectomy nor elevated by dexamethasone treatment. However, the relative amount of SR-associated CaM kinase II protein was increased 2.5- to 4-fold in dexamethasone-treated rats compared with control and adrenalectomized rats. Endogenous CaM kinase II activity, as judged from phosphorylation of ryanodine receptor, Ca²⁺-ATPase, and phospholamban protein, was also significantly higher (50-80% increase) in the dexamethasone-treated rats. The stimulatory effect of CaM kinase II activation on Ca²⁺ uptake activity of SR was significantly depressed after adrenalectomy and greatly enhanced after dexamethasone treatment. These findings identify the SR as a major target for glucocorticoid actions in the heart and implicate modification of the SR CaM kinase II system as a component of the mechanisms by which dexamethasone influences SR Ca²⁺-cycling and myocardial contraction.