This study investigated the Ca²⁺ cycling properties of sarcoplasmic reticulum (SR) in the right ventricle (RV) and left ventricle (LV) of normal rat myocardium. In addition, intracellular Ca²⁺ transients and contractile function were monitored in freshly isolated myocytes from RV and LV. Surprisingly, RV-SR displayed ~4-fold lower rates of ATP-energized Ca²⁺uptake in vitro, compared with LV-SR. The concentration of Ca²⁺ required for half-maximal activation of Ca²⁺ transport was ~2-fold higher in RV-SR. The lower Ca²⁺ sequestering activity of RV-SR was accompanied by a matching decrement in Ca²⁺-induced phosphoenzyme formation during the catalytic cycle of the Ca²⁺-pumping ATPase (SERCA2). Western immunoblotting analysis showed protein levels of Ca²⁺-ATPase and its inhibitor, phospholamban (PLN), to be only ~15% lower in RV-SR, compared with LV-SR. Co-immunoprecipitation experiments revealed that PLN-bound, functionally inert Ca²⁺-ATPase molecules in RV-SR greatly exceed (>50% difference) that in LV-SR. Endogenous Ca²⁺/calmodulin-dependent protein kinase (CaM kinase) mediated phosphorylation of SR substrates did not abolish the huge disparity in SR Ca²⁺-pump function between RV and LV. Intracellular Ca²⁺ transients, evoked by electrical field stimulation, were significantly prolonged in RV myocytes, compared to LV myocytes, mainly due to slow [Ca²⁺]_i decay. The slow [Ca²⁺]_i decay in RV, and consequent decrease in the speed of RV relaxation, may serve to promote temporal synchrony of the end of diastole in right and left ventricular chambers. The preponderance of functionally silent SR Ca²⁺-pumps in RV reflects a higher diastolic reserve required to protect and maintain RV function when faced with a sudden rise in afterload or resistance in the pulmonary circulation.