The peroxidative susceptibility in cultured rat hepatocytes of eicosapentaenoic acid (EPA) and other polyunsaturated fatty acids (PUFA) with different numbers of double bonds was examined. Lipid peroxidation was evaluated using a newly developed HPLC procedure which includes the determination of malondialdehyde (MDA). Following exposure to 0.25-1.0 mM EPA adsorbed to BSA (EPA-BSA), cultured hepatocytes produced MDA in the fatty acid concentration- and incubation time-dependent manner. The rate of MDA production by hepatocytes varied greatly with the degree of PUFA unsaturation, and ranked as follows: docosahexaenoic acid > EPA > arachidonic acid > α-linolenic acid = γ-linolenic acid > linoleic acid > oleic acid. Prolonged exposure of cultured hepatocytes to 1.0 mM EPA-BSA resulted in substantial leakage of LDH into the medium. The cell injury was associated with the loss of cellular GSH and protein thiol groups. Cotreatment of the EPA-supplemented hepatocytes with a GSH-depleting agent, diethylmaleate, promoted the cellular protein thiol loss and LDH leakage. An iron chelator, deferoxamine, and other antioxidants such as N,N-diphenyl- p-phenylenediamine and γ-tocopherol efficiently prevented MDA production and consequently LDH leakage in the EPA-supplemented hepatocytes. These results show that peroxidative deterioration in excess of GSH-dependent defense mechanisms may occur in hepatocytes loaded with highly peroxidizable fish oil PUFA.