Acetaminophen overdose causes massive hepatic failure via mechanisms involving glutathione depletion, oxidative stress, and mitochondrial dysfunction. The ultimate target of acetaminophen causing cell death remains uncertain, and the role of apoptosis in acetaminophen‐induced cell killing is still controversial. Our aim was to evaluate the mitochondrial permeability transition (MPT) as a key factor in acetaminophen‐induced necrotic and apoptotic killing of primary cultured mouse hepatocytes. After administration of 10 mmol/L acetaminophen, necrotic killing increased to more than 49% and 74%, respectively, after 6 and 16 hours. MPT inhibitors, cyclosporin A (CsA), and NIM811 temporarily decreased necrotic killing after 6 hours to 26%, but cytoprotection was lost after 16 hours. Confocal microscopy revealed mitochondrial depolarization and inner membrane permeabilization approximately 4.5 hours after acetaminophen administration. CsA delayed these changes, indicative of the MPT, to approximately 11 hours after acetaminophen administration. Apoptosis indicated by nuclear changes, terminal deoxynucleotidyl transferase‐mediated dUTP nick end labeling, and caspase‐3 activation also increased after acetaminophen administration. Fructose (20 mmol/L, an adenosine triphosphate–generating glycolytic substrate) plus glycine (5 mmol/L, a membrane stabilizing amino acid) prevented nearly all necrotic cell killing but paradoxically increased apoptosis from 37% to 59% after 16 hours. In the presence of fructose plus glycine, CsA decreased apoptosis and delayed but did not prevent the MPT. In conclusion, after acetaminophen a CsA‐sensitive MPT occurred after 3 to 6 hours followed by a CsA‐insensitive MPT 9 to 16 hours after acetaminophen. The MPT then induces ATP depletion–dependent necrosis or caspase‐dependent apoptosis as determined, in part, by ATP availability from glycolysis. (HEPATOLOGY 2004;40:1170–1179.)