Phosphorylation of sarcoplasmic reticulum (SR) Ca²⁺-cycling proteins by a membrane-associated Ca²⁺/calmodulin-dependent protein kinase II (CaM kinase II) is a well documented physiological mechanism for regulating transmembrane Ca²⁺ fluxes and cardiomyocyte contraction-relaxation cycle. The present study investigated the effects of L-thyroxine-induced hyperthyroidism on protein expression of SR CaM kinase II and its substrates, endogenous CaM kinase II-mediated SR protein phosphorylation, and SR Ca²⁺ pump function in the rabbit heart. Membrane vesicles enriched in junctional SR (JSR) or longitudinal SR (LSR) isolated from euthyroid and hyperthyroid rabbit hearts were utilized. Endogenous CaM kinase II-mediated phosphorylation of ryanodine receptor-Ca²⁺ release channel (RyR-CRC), Ca²⁺-ATPase and phospholamban (PLN) was significantly lower (30-70%) in JSR and LSR vesicles from hyperthyroid rabbit heart, compared to euthyroid. Western immunoblotting analysis revealed significantly higher levels of SERCA2 isoform of Ca²⁺-ATPase in JSR (~ 40%) but not in LSR from the hyperthyroid compared to euthyroid group. The maximal velocity of Ca²⁺ uptake (V_max) was significantly increased in JSR (130%) and LSR (50%) from hyperthyroid, compared to euthyroid, rabbit hearts. The apparent affinity of the Ca²⁺-ATPase for Ca²⁺ (K_0.5) did not differ between the two groups. The protein levels of PLN and CaM kinase II were significantly lower (30-40%) in JSR, LSR and ventricular tissue homogenates from the hyperthyroid rabbit heart. These findings demonstrate selective down regulation of the expression and function of CaM kinase II in the hyperthyroid rabbit heart in the face of upregulated expression and function of SERCA2 predominantly in the JSR compartment.