The ability of specific low molecular weight sulfhydryl compounds to inhibit the myoglobin-H2O2 peroxidation of uric acid and arachidonic acid was investigated. alpha-Mercaptopropionyl glycine, N-acetylcysteine, and reduced glutathione inhibited both the oxymyoglobin and metmyoglobin H2O2-mediated peroxidation of uric acid in a dose-dependent manner. The IC50 for each drug ranged between 20 to 100 microM and was dependent on the presence of a reduced sulfhydryl group since neither oxidized glutathione nor methionine effectively blocked uric acid peroxidation. Similar inhibition of oxymyoglobin and metmyoglobin H2O2-mediated peroxidation of arachidonic acid was also observed with alpha-mercaptopropionyl glycine, reduced glutathione, and cysteine. Under conditions of this assay, the ferrous form of myoglobin and H2O2 produced approximately three times the amount of formaldehyde from dimethylsulfoxide than ferric myoglobin (metmyoglobin) and H2O2. However, metmyoglobin and H2O2 were more effective than either oxymyoglobin and deoxymyoglobin in mediating arachidonic acid peroxidation. Further, neither mannitol nor benzoic acid (known scavengers of .OH) effectively blocked myoglobin H2O2-induced peroxidation of either uric acid or arachidonic acid. Visible absorption spectra of oxymyoglobin and metmyoglobin after incubation with H2O2 indicates the formation of a relatively stable ferriperoxide derivative of myoglobin. The formation of the ferriperoxide myoglobin derivative was partially inhibited by the addition of reduced sulfhydryl compounds. These data are consistent with the hypothesis that during reperfusion injury of the ischemic myocardium, the phagocytic cell or intracellular-derived H2O2 may react with myoglobin and initiate peroxidation reactions independent of .OH formation leading to cell injury. The cardioprotective effects of alpha-mercaptopropionyl glycine and other sulfhydryl-containing compounds during reperfusion injury may be attributed, at least in part, to their ability to inhibit myoglobin-H2O2-mediated peroxidation reactions.