To study the biochemical and toxicological properties of cytochrome P450 2E1 (CYP2E1), an adenovirus containing human CYP2E1 cDNA (Ad-CYP2E1) was constructed and was shown to successfully mediate the overexpression of CYP2E1 in HepG2 cells. Acetaminophen (APAP) toxicity to HepG2 cells infected with Ad-CYP2E1 was characterized as a preliminary proof of principle experiment to validate the functionality of the CYP2E1 adenovirus. Compared with cells infected with Ad-LacZ, HepG2 cells infected with Ad-CYP2E1 were more sensitive to APAP induced necrosis and apoptosis when the cells were depleted of intracellular reduced glutathione (GSH). The APAP cytotoxicity was dependent on both the concentration of APAP and the multiplicity of infection of the Ad-CYP2E1 virus. Apoptosis induced by APAP in HepG2 cells overexpressing CYP2E1 was caspase dependent and could be inhibited by the pan-caspase inhibitor Z-VAD-fmk. After treatment with APAP, mitochondrial membrane potential was dramatically decreased in the CYP2E1-expressing cells. APAP protein adducts were elevated in HepG2 cells infected with Ad-CYP2E1 compared with that in cells infected with Ad-LacZ; two bands around 90 KD were found only in the CYP2E1-expressing cells. These results demonstrate that adenovirus-mediated overexpression of human CYP2E1 activates APAP to reactive metabolites which damage mitochondria, form protein adducts, and result in toxicity to HepG2 cells. The Ad-CYP2E1 may be useful for studies designed to investigate the role of CYP2E1 in APAP and alcoholic liver injury and to further characterize the actions and effects of CYP2E1.