This study was concerned with the role of Cu and Cu-MT (metallothionein) in oxidative stress. Because hepatic Cu and Cu-MT concentrations are known to be high in the 3-day-old guinea pigs but decline to low adult levels by 7 days of life, the hepatotoxicity of ferric nitrilotriacetate (FeNTA) in the developing guinea pig was used as the experimental model in the present study. Results of this study showed that the hepatotoxic response to FeNTA (3.5 mg Fe /kg i.p.) as measured by elevation in serum aspartate aminotransferase activity, increase in lipid peroxidation, decrease in reduced glutathione/oxidized glutathione ratio and histopathological changes was higher in 3-day-old than in 7-day-old and adult guinea pigs. Furthermore, pretreatment of 7-day-old guinea pigs with cupric sulfate (0.5 mg Cu++/kg i.p.) increased hepatic Cu and Cu-MT levels and enhanced susceptibility to FeNTA. FeNTA treatment resulted in the oxidation of MT thiolates and reduction in the metal binding capacity and Cu content of MT in the 3-day-old and Cu-pretreated 7-day-old animals, providing evidence for the interaction between Cu-MT and cellular oxidants. In vitro study with FeNTA and hepatic microsomes revealed no age-related differences in microsomal lipid peroxidation; however, this parameter was stimulated in the presence of control or heat-treated cytosols isolated from 3-day-old but not those of 7-day-old animals. These observations were consistent with the involvement of Cu-MT, a heat-stable metalloprotein, in the sensitization of hepatic tissues to oxidative injury in the 3-day-old animal. Moreover, in vitro study involving the use of D-penicillamine, a Cu chelating agent, showed that the sensitization effect of Cu-MT was mediated by Cu ions. The results of this study suggest that Cu-MT may have a prooxidative property and tissues with high Cu-MT levels may be particularly susceptible to oxidative stress.