Background: Calcium overload and free radical mediated damage in the mitochondria is the most important pathological changes associated with myocardial ischemic-reperfusion injury. The verapamil post-treatment has been previously reported to prevent reperfusion-induced myocardial injury but functional recovery may be delayed due to the drug's inherent direct myocardial depression effect. In the present study the effect of verapamil on mitochondrial enzymes and sarcoplasmic ATPase system was examined during the myocardial preconditioning and ischemic reperfusion in rat heart. Methods: Four groups of isolated rat hearts were perfused with KH buffer in a retrograde manner by Lagendroff apparatus. A time controlled ischemia, ischemic reperfusion and classical precondition was produced by restoring the flow of KH buffer in ischemic rat heart. The hearts were then processed to isolate the mitochondria and sarcoplasmic reticulum for the biochemical estimation. Results: Mitochondrial enzyme and sarcoplasmic ATPase activities were diminished in the ischemic period and further decreased during reperfusion. However, preconditioning the rat heart before the insult of ischemia and reperfusion improved the enzyme activities. The preconditioning procedure consisted of 4 cycles of 4 min. short ischemic periods followed by 4 min. KH buffer perfusion applied before 30 min. global ischemia and reperfusion caused an improvement in the mitochondrial enzyme activities. On the other hand, sarcoplasmic ATPase enzymes required a precondition procedure of 7 cycles of 2 min. short ischemic periods followed by 2 min. reperfusion. The activities of the above enzymes were improved further when verapamil was administered before the insult of ischemic reperfusion. Conclusion: This study shows the beneficial effect of classical preconditioning with verapamil.