Under the 2005 U.S. EPA Guidelines for Carcinogen Risk Assessment (1), evaluations of carcinogens rely on mode of action data to better inform dose response assessments. A reassessment of carbon tetrachloride, a model hepatotoxicant and carcinogen, provides an opportunity to incorporate into the assessment biologically relevant mode of action data on its carcinogenesis. Mechanistic studies provide evidence that metabolism of carbon tetrachloride via CYP2E1 to highly reactive free radical metabolites plays a critical role in the postulated mode of action. The primary metabolites, trichloromethyl and trichloromethyl peroxy free radicals, are highly reactive and are capable of covalently binding locally to cellular macromolecules, with preference for fatty acids from membrane phospholipids. The free radicals initiate lipid peroxidation by attacking polyunsaturated fatty acids in membranes, setting off a free radical chain reaction sequence. Lipid peroxidation is known to cause membrane disruption, resulting in the loss of membrane integrity and leakage of microsomal enzymes. By-products of lipid peroxidation include reactive aldehydes that can form protein and DNA adducts and may contribute to hepatotoxicity and carcinogenicity, respectively. Natural antioxidants, including glutathione, are capable of quenching the lipid peroxidation reaction. When glutathione and other antioxidants are depleted, however, opportunities for lipid peroxidation are enhanced. Weakened cellular membranes allow sufficient leakage of calcium into the cytosol to disrupt intracellular calcium homeostasis. High calcium levels in the cytosol activate calcium-dependent proteases and phospholipases that further increase the breakdown of the membranes. Similarly, the increase in intracellular calcium can activate endonucleases that can cause chromosomal damage and also contribute to cell death. Sustained cell regeneration and proliferation following cell death may increase the likelihood of unrepaired spontaneous, lipid peroxidation- or endonuclease-derived mutations that can lead to cancer. Based on this body of scientific evidence, doses that do not cause sustained cytotoxicity and regenerative cell proliferation would subsequently be protective of liver tumors if this is the primary mode of action. To fulfill the mode of action framework, additional research may be necessary to determine alternative mode(s) of action for liver tumors formed via carbon tetrachloride exposure.