The H2O2 generated by menadione kills cultured hepatocytes by a mechanism that depends in large part on a cellular source of ferric iron. Chelation of this iron by deferoxamine reduced by two-thirds the number of dead cells without any effect on the loss of 30% of total protein thiols, the formation of protein mixed disulfides, or the accumulation of oxidized glutathione (GSSG). The loss of protein thiols was accounted for by the formation of glutathione mixed disulfides from GSSG and the arylation of protein nucleophiles by menadione. Nevertheless, such a loss occurred despite the chelation of cellular iron and a substantial reduction in the extent of cell killing. With the H2O2 generated by glucose oxidase, lipid peroxidation and a loss of 40% of the total protein thiols accompanied the cell killing within 1 hr. Deferoxamine, superoxide dismutase and the antioxidant N,N'-diphenyl phenylenediamine (DPPD) prevented the cell killing and two-thirds of the loss of protein thiols. Peroxidation of liver microsomes in vitro with ADP:Fe3+ similarly depleted protein thiols, an effect that was prevented by DPPD. The supernatant fraction from the peroxidation assay depleted the protein thiols of cultured hepatocytes without an effect on viability. Thus, lipid peroxidation accounted for the major part of the loss of protein thiols with glucose oxidase. The 10-15% decrement in protein thiols after 1 hr that occurred in the absence of cell killing reflected the formation of glutathione mixed disulfides. Finally, in the presence of DPPD, glucose oxidase killed 75% of the cells between 1 and 3 hr without any further change in protein thiols. Thus, under the conditions studied, the depletion of protein thiols by the three mechanisms, namely lipid peroxidation, formation of glutathione mixed disulfides, and arylation, does not necessarily have a causal relationship to the killing of cultured hepatocytes.