Ca(2+) overload and free-radical injury are two mutually non-exclusive phenomena suggested to cause myocardial ischemia-reperfusion (IR)-induced contractile dysfunction; however, the mechanisms underlying their effects are not clear. One possible mechanism is the proteolytic modification of proteins by Ca(2+)-dependent proteases, such as calpains, which are activated during Ca(2+) overload that occurs in IR. The sarcoplasmic reticulum (SR) plays a central role in mediating cardiac contractility and therefore any impairment in SR function will induce cardiac contractile dysfunction. We therefore investigated the possibility whether SR proteins were the target for calpain action in IR. Langendorff-perfused rat hearts were subjected to IR in the presence and absence of leupeptin, a calpain inhibitor and the effects of calpain inhibition was examined on cardiac performance, SR function, and its regulation by protein phosphorylation as well as expression of SR Ca(2+)-cycling and -regulatory proteins. Our results show a depression in cardiac contractile function and activation of calpain during IR. Treatment with leupeptin recovered cardiac contractile function and attenuated calpain activity in IR hearts. The cardioprotection observed upon leupeptin treatment was associated with improved SR function and regulation. The recovery in SR function and regulation was consistent with prevention of IR-induced decrease in the expression of key SR Ca(2+)-handling and -regulatory proteins. Our results suggest that a downregulation of SR proteins by calpain may be a mechanism by which Ca(2+) overload causes cardiac contractile dysfunction during IR.