Disruption of lysosomal membranes is one of the earliest changes seen in the ischemic myocardium and a calcium-calmodulin-dependent process may be involved in irreversible myocardial injury. This study was designed to determine the effect of trifluoperazine (TFP), a calmodulin antagonist, on the isolated rat heart. To assess the cardioprotective effect of TFP, changes in the distribution of lysosomal enzymes in myocardial cells and post-ischemic myocardial recovery were studied. Experimental groups (n=6 hearts per group) were: Group I, infusion of 20°C Krebs-Henseleit bicarbonate buffer (KHB) every 30 min during ischemia; Group II, infusion of KHB containing TFP (2.5×10(-6)M/L); Group III, infusion of St. Thomas' Hospital cardioplegic solution (ST); Group IV, infusion of ST containing TFP (2.5×10(-6)M/L). Isolated perfused rat hearts were ischemic for 2 h at 20°C and were reperfused at 37°C for 30 min. Functional recovery after ischemia and reperfusion was measured by developed pressure. Addition of TFP in Group II hearts prevented leakage of lysosomal enzymes (N-acety1-β-glucosaminidase and Cathepsin D) compared with Group I (p<0.05, respectively). The activities of lysosomal enzymes in cytosol were lower in Group IV than in Group III, though the difference was not statistically significant. Hearts receiving TFP (Group II) showed improved recovery of developed pressure at 15 min and 30 min after reperfusion when compared with Group I (p<0.01, respectively). These results suggest that TFP reduces ischemia/reperfusion injury and calcium-calmodulin-dependent enzymes may play an important role in the development of cellular damage in the myocardium during hypothermic ischemia.