This study of acetaminophen (AAP) hepatotoxicity examined whether some aspects of the highly integrated process of drug-induced toxicity involves apoptosis, in addition to necrosis in vivo; and if so, whether cholesteryl hemisuccinate (CS) pretreatment would selectively interfere with apoptotic or necrotic liver cell death. We have previously demonstrated that CS preexposure in vivo, protects hepatocellular necrosis and necrosis-related events induced by carbon tetrachloride (CCl4) administration. Our study demonstrates that administration of hepatotoxic doses of AAP (350-500 mg/kg, i.p.) to ICR mice (CD-1) results in severe liver injury leading to cell death both by necrosis and apoptosis. AAP-induced cell death was preceded by massive elevation in serum alanine aminotransferase coupled with rapid loss of large genomic DNA (2-24 hr), fragmentation of DNA in the form of a ladder (2-24 hr), apoptotic nuclear condensation at early hours (2-6 hr) followed by massive fragmentation and margination of heterochromatin at later (6-24) hours and a near total loss of glycogen in pericentral areas. Although CS (100 mg/kg, i.p.) alone had no noticeable biochemical or morphological effects, its administration before AAP (350-500 mg/kg, i.p.) abrogated histological and biochemical diagnostics of both apoptosis and necrosis. These include near total absence of loss of large genomic DNA and glycogen, and dramatic protection from escalating levels of liver injury. CS pretreatment also arrested AAP-induced ultrastructural changes typical of both apoptosis and necrosis. Histopathological examination of periodic acid-Schiff stained liver sections mirrored the biochemical and ultrastructural findings. In conclusion, this study for the first time establishes that apoptosis, in addition to necrosis, significantly contributes to AAP hepatotoxicity in vivo, and preexposure of mice to CS prevents AAP-induced hepatic apoptosis and necrosis.