Although cardiac endogenous antioxidants have been reported to be oxidized and decreased by ischemia-reperfusion, little is known whether the changes in these antioxidants are correlated with each other in a systematic relationship. In this study, isolated rat hearts were subjected to various periods of ischemia-reperfusion using the Langendorff method, and the content and/or redox status of tissue antioxidants were analyzed. Significant losses in the tissue hydrophilic antioxidants, ascorbate, and glutathione were observed. These losses were dependent on the duration of the reperfusion period (between 0-40 min) but not of ischemia (20-60 min). Marked increases of dehydroascorbate and glutathione disulfide, the oxidized forms of ascorbate and glutathione, respectively, were found during reperfusion, but these changes were not observed during ischemia. These findings indicate that the tissue hydrophilic antioxidants are easily oxidized and may be the first line of antioxidant defenses during reperfusion. Lipophilic antioxidants, like ubiquinol 9 and vitamin E, were not decreased during ischemia-reperfusion using regular buffer; however, if oxidative stress was induced by addition of H2O2 to the buffer solution during reperfusion after 20 min of ischemia, decreases in both the hydrophilic and hydrophobic antioxidants were noticeable. With 100 microM H2O2, the tissue antioxidant decreases were ubiquinol 9 (39%), vitamin E (3%), glutathione (44%) and ascorbate (58%). Only with 500 microM H2O2 treatment were marked decreases in tissue vitamin E (65%) observed; this was associated with almost complete depletion of tissue ubiquinol 9 (95%). These results suggest that prior to the consumption of vitamin E, other antioxidants are depleted and that vitamin E may serve as the ultimate antioxidant, protecting the integrity of cellular membranes. Thus, in this work, cardiac antioxidants were demonstrated to change in a systematically organized relationship under ischemia-reperfusion. This graded utilization of antioxidants supports the redox based antioxidant network concept, found to be present in other biological systems.