Ethoxycoumarin is metabolized by mixed-function oxidation to give 7-hydroxycoumarin (umbelliferone) and acetaldehyde, without formation of an intermediate electrophile. Ethoxycoumarin was found, nevertheless, to injure cultured rat hepatocytes. Male hepatocytes were more sensitive than female to ethoxycoumarin. Phenobarbital increased cell killing, and SKF 525A, an inhibitor of ethoxycoumarin metabolism, prevented it. Neither umbelliferone nor acetaldehyde were toxic. Cellular glutathione decreased and oxidized glutathione (GSSG) accumulated in the culture medium. Sulfhydryl reagents prevented the cell killing without inhibiting metabolism. Lipid peroxidation was detected prior to evidence of cell death, and the antioxidant N,N'-diphenyl-phenylenediamine prevented both the lipid peroxidation and cell killing without inhibiting metabolism. Inhibition of glutathione reductase with 1,3-bis(chloroethyl)-1-nitrosourea potentiated the cell killing without increasing metabolism. Pretreatment of the cells with the ferric iron chelator deferoxamine reduced cell killing, again without inhibiting metabolism. Ferric chloride restored the sensitivity of deferoxamine-pretreated hepatocytes to ethoxycoumarin. These data define a new experimental model in which lethal liver cell injury is dependent on the metabolism of ethoxycoumarin but unrelated to its two known metabolites. An oxidative stress accompanying the cytochrome P-450-dependent metabolism of ethoxycoumarin is proposed as the mechanism coupling metabolism to lethal cell injury.