Alterations in the intracellular Ca²⁺ handling in cardiomyocytes may underlie the cardiac dysfunction observed in the ovarian sex hormones-deprived condition. To test the hypothesis that ovarian sex hormones had a significant role in the cardiac intracellular Ca²⁺ mobilization, the sarcoplasmic reticulum (SR) Ca²⁺ uptake and the SR Ca²⁺-ATPase (SERCA) activities were determined in 10-wk ovariectomized rat hearts. Using left ventricular homogenate preparations, a significant suppression of maximum SR Ca²⁺-uptake activity but with an increase in the SR Ca²⁺ responsiveness was demonstrated in ovariectomized hearts. In parallel measurements of SERCA activity in SR-enriched membrane preparations from ovariectomized heart, a suppressed maximum SERCA activity with a leftward shift in the relationship between pCa (-log molar free Ca²⁺ concentration) and SERCA activity was also detected. A significant downregulation of SERCA proteins and reduction in the SERCA mRNA level were observed in association with suppressed maximum SERCA activity. While there were no changes in the total phospholamban and the phospho-Ser¹⁶ phospholamban levels, a decrease in the phospho-Thr¹⁷ phospholamban as well as an increase in the supra-inhibitory, monomeric, form of phospholamban stoichiometry were found. Estrogen and progesterone supplementations were equally effective in preventing changes in ovariectomized hearts. Our data showed for the first time that female sex hormones played an important role in the regulation of the cardiac SR Ca²⁺ uptake. Under the hormone-deficient condition, there was an adaptive response of the SERCA that escaped from the regulatory effect of phospholamban.