It is well known that activation of proteases in the lysosomes and cytosol is one of the mechanisms of ischemic injury. It might thus be beneficial to determine whether the addition of several clinically available protease inhibitors to a cardioplegic solution can improve its protective ability. Using an isolated working rat heart preparation, the effects of several protease inhibitors (serine protease inhibitors; nafamostat mesilate and gabexate mesilate, a thiol-protease inhibitor; NCO-700; and a urinary trypsin inhibitor, urinastatin) on the postischemic recovery of function and enzyme leakage were investigated in this study. These protease inhibitors were added to either the cardioplegic solution or reperfusion solution. The addition of each of the protease inhibitors, except urinastatin, to the cardioplegic solution improved the postischemic recovery of function and reduced enzyme leakage. The dose-response characteristics of these three protease inhibitors were bell shaped, and the optimal concentrations of nafamostat mesilate, gabexate mesilate, and NCO-700 were 5 µM, 100 µM, and 20 µM, respectively. In contrast to the results of the preischemic treatment study, the addition of any of the protease inhibitors to the perfusion medium during Langendorff reperfusion failed to improve the postischemic recovery of function and to reduce enzyme leakage. Surprisingly, the addition of NCO-700 to the reperfusion solution at a concentration of 5 µM or higher had rather harmful effects on both functional recovery and enzyme leakage. These findings suggest that serine and thiol proteases may play an important role in myocardial injury during ischemia, but not necessarily during reperfusion.