Acetaminophen is activated metabolically to yield reactive species that bind covalently to liver cell macromolecules. The extent of covalent binding correlates with the occurrence and severity of hepatic necrosis. We reported previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)] that active Ca2+ accumulation of isolated liver plasma membranes is decreased 60-75% after a hepatotoxic dose of acetaminophen in vivo. We now report that the protein of isolated liver plasma membranes was substantially labeled with drug metabolites after administration of [3H]acetaminophen. There was no increase in passive membrane permeability that might cause diminished Ca2+ accumulation. Intravesicular volume and relative purity of the vesicle preparations after acetaminophen were not different from controls. However, (Ca2+,Mg2+)-ATPase, a possible biochemical expression of the Ca2+ pump, was decreased 31% (P less than 0.025) after acetaminophen treatment. ATPase activity in both control and treated groups was enhanced by isolating membranes in the presence of 5 mM reduced glutathione (GSH), but the effects of drug treatment were not reversed. A similar effect of GSH on Ca2+ accumulation was observed previously [J. O. Tsokos-Kuhn, E. L. Todd, J. B. McMillin-Wood and J. R. Mitchell, Molec. Pharmac. 28, 56 (1985)]. These data are consistent with a hypothesis wherein alkylation of membrane proteins by reactive acetaminophen metabolites is a factor in the onset of hepatic necrosis after acetaminophen. They are not consistent with an oxidative stress hypothesis where thiol S-thiolation of membrane components is postulated to produce altered membrane permeability or thiol-reversible alterations in membrane protein structure and enzymatic function.