The present experiments were performed to confirm the hypothesis that xanthine oxidase[XOD], as a source and mechanism of oxygen radical production, plays an important role in the genesis of the reperfusion injury of ischemic myocardium. The experimental ischemic-reperfusion injury was induced in isolated, Langendorff preparations of rat hearts by 60 min. Of global ischemia with aortic clamping followed by 20 min. of reperfusion with oxygenated Krebs-Henseleit solution[pH 7.4, 37*C]. The results were as follows: 1. The releases of creatine phosphokinase and a lipid peroxidation product, malondialdehyde[MDA] into the coronary effluent were abruptly increased upon reperfusion of ischemic hearts. The increases of the enzyme and MDA were suppressed significantly in the hearts removed from rats pretreated with allopurinol, a specific XOD inhibitor[20mg/kg, oral, 24 hrs and 2 hrs before study]. This effect of allopurinol was comparable to that of oxygen radical scavengers, superoxide dismutase[5, 000U] and catalase[12, 500 U]. 2. The increased SOD-inhibitable reduction of ferricytochrome C, which was infused to the hearts starting with reperfusion, was significantly suppressed in allopurinol pretreated hearts. 3. Activities of myocardial XOD were compared in the normal control hearts and the ischemic ones. Total enzyme activities were not different in both hearts. However, comparing with the control, the ischemic ones showed higher activity in 0-form and lower activities in D-form and D/O-form. 4. In the ischemic hearts, phenylmethylsulfonyl fluoride, a serine protease inhibitor, prevented significantly the increase of 0-form and the decreases of D and D/O-form, while thiol reagents did not affect the changes of the enzyme. 5. The increase of 0-form and the decreases of D and D/0-form were not significant in both calcium-free perfused and pimozide, a calmodulin inhibitor, treated ischemic hearts. 6. The SOD-inhibitable reduction of ferricytochrome C were suppressed by PMSF and pimozide treatment as well as by calcium-free perfusion. It is suggested from these results that in the ischemic rat myocardium, xanthine oxidase is converted to oxygen radical producing 0-form by calcium, calmodulin-dependent proteolysis and plays a contributing role in the genesis of ischemic-reperfusion injury by producing oxygen free radicals.