Redox properties of metallothioneins (MTs) and Cu in the cytosol from Long–Evans Cinnamon (LEC) rat livers 13 weeks after birth were investigated. MTs from LEC rat livers contain 8 g atoms of Cu and 1 g atom of Zn per mole of protein (Cu(I)₈-MTs). Titration of Cu(I)₈-MTs with CuCl₂ indicates that Cu(I)₈-MTs were able to reduce further 2-g atoms of cupric ions per mole MTs as bound form. Hg²⁺-induced hydroxyl radical generation from Cu(I)₈-MTs was demonstrated by ESR using the spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). The intensity of DMPO-OH signal from Cu-loaded MTs was increased with the increasing number of Cu in MTs. The used cytosol fraction contained 1.37 mM total Cu and 5 mM DTNB titrable-SH groups has a potential to reduce 2 mM CuCl₂. No ESR signal due to Cu²⁺ was also detected with LEC rat liver cytosol, whereas strong Cu²⁺ signal appeared by the addition of HgCl₂. The rate constants for the reaction of Cu(I)₈-MTs with superoxide and hydroxyl radicals were estimated to be 2 × 10⁶ and ≥10¹² M⁻¹s⁻¹, respectively, from competition kinetics. Cu²⁺-catalyzed oxidation of DNA was strongly inhibited both in the presence of Cu-unsaturated MTs and GSH. The results suggest that Cu(I)₈-MTs from LEC rat livers just before hepatitis still act as antioxidants.