To test the hypothesis that 5'-nucleotidase activity during ischemia is attenuated by oxygen-derived free radicals, we measured ischemia-induced reactive hyperemic flow, adenosine release, and 5'-nucleotidase activity in dogs (n=62). A 1-minute occlusion of the coronary artery caused reactive hyperemic flow (307±5 versus 92±1 ml 100 g−1min−1at baseline) with increased release of adenosine (14.4±1.4 versus 0.4±0.1 nmol. 100 g−1min−1at baseline). Superoxide dismutase augmented (p<0.001) both peak coronary blood flow (333±6 ml 100 g−1min−1) and repayment (436±12 versus 320±7 ml/100 g in the untreated group). Adenosine release during reperfusion was augmented (22.7±1.9 nmol 100 g−1min−1,p<0.001), and 8-phenyltheophylline completely abolished the enhanced reactive hyperemia. Enzymatic assay of 5'-nucleotidase activity revealed that the administration of superoxide dismutase increases ecto-5'-nucleotidase activity in ischemic myocardium. When an inhibitor of ecto-5'-nucleotidase, α,β-methyleneadenosine 5'-diphosphate, was administered, the effects of superoxide dismutase were completely abolished. Thus, we conclude that 1) the augmentation of reactive hyperemic flow caused by superoxide dismutase is attributed to the enhanced release of adenosine and 2) the enhanced release of adenosine over the untreated controls is attributed to the protection of ecto-5'-nucleotidase activity during ischemia.