CYP2E1, a member of the cytochrome P450 family, is induced by ethanol. CYP2E1 activates many hepatotoxins to their reactive toxic intermediate form, and generates reactive oxygen species (ROS) during its catalytic cycle. Induction of CYP2E1 plays an important role in ethanol-induced oxidant stress and ethanol toxicity. To study the biochemical and toxicological properties of CYP2E1, our laboratory developed a HepG2 cell line which constitutively expresses the human CYP2E1 form. These cells displayed elevated oxidative stress, loss of mitochondrial function and loss of viability when challenged with prooxidants such as ethanol, polyunsaturated fatty acids (PUFA) such as arachidonic acid, iron, or when depleted of the critical antioxidant glutathione, as compared with control HepG2 cells which do not express CYP2E1. In the sections below , protocols are described for use of these cell lines to assay for CYP2E1-dependent oxidant stress and toxicity. Methods are described as to how the cell lines were established and maintained, how CYP2E1 is assayed, how cellular viability, mitochondrial function and generation of oxidant stress are determined.