These experiments were performed to characterize the relationship between lipid peroxidation and hepatocyte viability in iron overload. Hepatocytes were isolated from rats with chronic dietary iron overload and the effects of in vitro iron chelation on lipid peroxidation, cell viability and ultrastructure were studied over a 4-hr incubation period. Cell viability was significantly reduced at 3 and 4 hr in iron-loaded hepatocytes compared with controls and was preceded by an increase in iron-dependent lipid peroxidation. Similarly, extensive degenerative ultrastructural changes were observed in iron-loaded hepatocytes compared with controls after 4 hr of incubation. In vitro iron chelation with either deferoxamine or apotransferrin protected against lipid peroxidation, loss of viability and ultrastructural damage in iron-loaded hepatocytes. The addition of an antioxidant, α-tocopherol, also protected against lipid peroxidation and preserved cell viability over a 4-hr incubation. The protective effects of iron chelators and α-tocopherol support a strong association between iron-dependent lipid peroxidation and hepatocellular injury in iron overload. (HEPATOLOGY 1990;12:31–39).