We hypothesize that activation of heat shock protein 70 (HSP70) by preconditioning, which is known to confer delayed cardioprotection, attenuates the impaired handling of Ca²⁺ at multiple sites. To test the hypothesis, we determined how the ryanodine receptor (RyR), sarcoplasmic reticulum Ca²⁺-ATPase (SERCA) and Na⁺-Ca²⁺ exchange (NCX) handled Ca²⁺ in rat ventricular myocytes preconditioned with a kappa-opioid receptor agonist, U50,488H (UP), followed by blockade of HSP70 with a selective antisense oligonucleotides and subsequently subjected to simulated ischemia. We determined: 1) the Ca²⁺ transients induced by electrical stimulation and caffeine, which provide the overall picture on Ca²⁺ homeostasis; 2) expression of RyR, SERCA and NCX, and 3) Ca²⁺ fluxes via NCX by the use of ⁴⁵Ca²⁺ in the rat ventricular myocyte. We found that UP increased the activity of RyR, SERCA and NCX, and the expression of RyR and SERCA. These led to increases in release of Ca²⁺ from sarcoplasmic reticulum via RyR and in removal of Ca²⁺ from cytoplasm by re-uptake of Ca²⁺ to SR via SERCA and by extrusion of Ca²⁺ out of the cell via NCX. UP also reduced mitochondrial Ca²⁺ accumulation. All the effects of UP were either abolished or significantly attenuated by blockade of HSP70 synthesis with a selective antisense oligonucleotides. The results are evidence that activation of HSP70 by preconditioning improves the ischemia-impaired Ca²⁺ homeostasis at multiple sites in the heart, which may be responsible, at least partly, for attenuated Ca²⁺ overload, improved recovery in contractile function and cardioprotection.