To determine the importance of intracellular reactive oxygen metabolites in ischaemia and reperfusion, rat hearts were depleted of glutathione, a major intracellular antioxidant, prior to total global or low flow ischaemia. Chronic pretreatment of rats with up to 7 mmol·kg-1L-buthionine-S,R-sulphoximine over a period of 96 hours resulted in 85% depletion of total myocardial glutathione. Subsequent recovery of glutathione after treatment with the sulphoximine was slow, reaching only 54% of control levels, 96 hours after the final dose. Depletion of myocardial glutathione by 70% did not increase the metabolic consequences of either total global or low flow ischaemia in the isolated perfused heart, as determined by 31P NMR spectroscopy and high performance liquid chromatographic analysis of purine release. During low flow ischaemia glutathione depletion caused a significant reduction in purine release. On reperfusion, functional recovery was depressed compared to controls. Hearts depleted of glutathione also showed a decrease in developed tension during normoxic perfusion prior to low flow ischaemia. There was no difference between the two groups before or after total global ischaemia. These results do not support the hypothesis of free radical mediated reperfusion injury; however, the method described here for the depletion of glutathione should prove a useful tool for further investigation into the role of glutathione in cardiac metabolism and function.