Disulfiram is a potent antioxidant that prevented the peroxidation of microsomal phospholipids induced by ADP/Fe3+ at concentrations as low as 1 microM. However, disulfiram had a biphasic action when used to assess the role of lipid peroxidation in the killing of cultured hepatocytes by an acute oxidative stress. At a relatively low concentration (10 microM), the antioxidant activity of disulfiram predominated, and there was protection against the killing of the hepatocytes by allyl alcohol, tert-butyl hydroperoxide, hydrogen peroxide, and diethyl maleate. As the concentration of disulfiram was increased above 10 microM, the extent of protection progressively decreased. Thus, with higher concentrations of disulfiram, there was a second action whose consequence is to obscure the protective effect of the lower doses. With the agents studied, this additional and as yet undefined action of disulfiram leads to the killing of the hepatocytes by a mechanism that is unrelated to the peroxidation of lipids. This biphasic action of disulfiram must be appreciated in any attempt to use this compound to assess the role of lipid peroxidation in toxic cell injury.