Our previous study has demonstrated that ovariectomy (Ovx) significantly increased the left ventricular developed pressure (LVDP) and ± dp/dtmax in the isolated perfused rat heart and the effects were reversed by female sex hormone replacemen. In the present investigation we studied the effects of Ovx for 6 weeks on Ca²⁺ homeostasis, that determines the contractile function. Particular emphasis was given to Ca²⁺ handling by ryanodine receptor (RyR) and Na⁺-Ca²⁺ exchange (NCX). ⁴⁵Ca²⁺ fluxes via the RyR, NCX and Ca²⁺ ATPase (SERCA) were compared to their expression in myocytes from Ovx rats with and without estrogen replacement. Further, we correlated the handling of Ca²⁺ by these Ca²⁺ handling proteins with the overall Ca²⁺ homeostasis by determining the Ca²⁺ transients induced by electrical stimulation and caffeine, which reveals the dynamic changes of [Ca²⁺]_i in the heart. In addition, we determined the expression and contribution of protein kinase A (PKA) to the regulation of the aforementioned Ca²⁺ handling proteins in Ovx rats. It was found that after Ovx there were i) increased Ca²⁺ fluxes via RyR and NCX, which were reversed not only by estrogen replacement, but more importantly by blockade of PKA, ii) an increased expression of PKA and RyR; iii) no increase in expression of NCX and SERCA. We suggest that hyperactivities of RyR and NCX are a result of up-regulation of PKA. The increased release of Ca²⁺ through RyR and removal of Ca²⁺ by NCX are believed to be responsible for the greater contractility and faster relaxation after Ovx.