O pen-chest dogs (total number used, 117) underwent 10 5-min coronary occlusions (O) interspersed with 10 min of reperfusion (R). When systolic thickening fraction was measured 9 min after each R, the first O-R cycle was found to cause the largest decrement, with only a slight additional loss during the next four cycles and no further loss during the last five cycles (group IV), suggesting that the first few episodes of ischemia preconditioned the myocardium against the stunning induced by the last five episodes. However, different results were obtained when the total deficit of wall thickening during the final 4-h R interval was measured. The total deficit was similar after one and three 5-min O (groups V and VI, respectively), indicating that the first ischemic episode did precondition against the next two episodes; however, it was approximately 2.5-fold greater after 10 O (group IV) than after 3, indicating that the first 3 episodes failed to precondition against the next 7. Thus, at some point between the 4th and 10th O, the preconditioning effect was lost and recurrent ischemic episodes started to have a cumulative effect. Measurements of free radicals with alpha-phenyl N-tert-butyl nitrone (PBN) demonstrated a burst of free radical generation immediately after the 1st, 5th, and 10th R (group VIII). The total cumulative release of PBN adducts during the initial 5 min of reflow was 58% less after the 5th R than after the 1st (P < 0.05) but did not differ significantly between the 1st and 10th R. When administered throughout the 10 O-R cycles, the .OH scavenger mercaptopropionyl glycine significantly enhanced the recovery of function (group I) and markedly suppressed the formation of free radicals (group VII). However, the beneficial effects of mercaptopropionyl glycine were completely, or largely, lost if the drug was discontinued after the first five (group II) or eight (group III) O-R cycles, respectively, implying that (a) the oxidative stress associated with the last five, or even two, cycles was sufficient to cause severe postischemic dysfunction, and (b) the cumulative injury caused by repetitive ischemic episodes is mediated by recurrent oxidative stress. This study provides direct in vivo evidence that oxygen radicals play an important role in the pathogenesis of myocardial stunning after repetitive ischemia, and implicates .OH as a primary culprit. Taken together, the data indicate that recurrent brief ischemic episodes result in recurrent bouts of oxyradical-mediated injury that have a cumulative effect on contractility, a situation that could lead to protracted or even chronic myocardial stunning.