The toxicity of acetaminophen in freshly isolated hamster hepatocytes was investigated. Cells exposed to 2.5 mM acetaminophen for 90 min, followed by washing to completely remove unbound acetaminophen, and resuspension in fresh buffer, showed a dramatic decrease in viability over the ensuing 4.5 hr by which time only 4% of the cells could still exclude trypan blue. During the initial 90-min incubation, there was a substantial depletion of glutathione, to 19% of control values, covalent binding of [14C]acetaminophen to cellular proteins, and evidence of morphological changes consistent with some disturbance of the plasma membrane. During subsequent incubation of these cells, covalent binding did not change nor did lipid peroxidation, despite the decrease in viability that occurred. Subsequent incubation of cells exposed to acetaminophen for 90 min in buffer containing 1.5 mM dithiothreitol (DTT), a disulfide-reducing agent, largely prevented the decrease in cell viability and reversed the morphological changes that occurred during the first 90-min incubation. However, there was no change in lipid peroxidation, glutathione content, or covalent binding. It is concluded that acetaminophen interacted with some critical target in the cell, and that this left unchecked, led eventually to the death of the cell. DTT prevented and reversed this effect. The toxicity of acetaminophen, and its reversal by DTT, appear independent of either covalent binding of acetaminophen or lipid peroxidation. In addition, the effect of DTT was independent of the concentration of glutathione, most probably acting by directly reducing oxidized SH-groups in critical enzymes, possibly membrane-bound ATP-dependent Ca2+ translocases.