Reperfusion of an isolated mammalian heart with a calcium-containing solution after a brief calcium-free perfusion results in irreversible cell damage: the calcium paradox. We investigated whether the calcium paradox is associated with oxidative damage. We measured the tissue changes of glutathione status and the release of oxidized glutathione from isolated perfused rabbit hearts as indicators of cellular oxidative events. After 10 min of calcium-free perfusion, tissue content of reduced (GSH) and oxidized (GSSG) glutathione, and of protein and non-protein sulfhydryl groups were not significantly different from control values. Restoration of the calcium concentration resulted in an immediate and massive release of GSH and a depletion of tissue content of GSH, GSSG, and non-protein sulfhydryl groups. However, only a minimal release of GSSG into the coronary effluent was observed. In addition, the characteristic features of the calcium paradox were present: development of an irreversible contracture and massive release of creatine kinase. The calcium paradox did not lead to a decrease of the tissue content of protein sulfhydryl groups. These observations indicate that the calcium paradox is not associated with oxidative damage.