Reactions of LEC (Long-Evans rats with a cinnamonlike coat color) rat liver Cu(I)-metallothioneins (MTs) with HgCl2 or K3Fe(CN)6 were investigated by ESR spectroscopy and generation of hydroxyl radicals was demonstrated using the ESR spin trap, 5,5-dimethyl-1-pyrroline N-oxide (DMPO). When Cu(I)-MTs were incubated with more than one equivalent mole HgCl2 or K3Fe(CN)6 to Cu+ bound to MTs, strong signals due to Cu2+ appeared. ESR spectra, which were a combination of the DMPO-OH adduct signal and a six-line signal, were observed in the reaction of Cu(I)-MTs with HgCl2, whereas no oxygen radical signal was seen with K3Fe(CN)6. The DMPO-OH signal intensity was greater in the presence of SOD while the signal disappeared in the presence of catalase. The results suggest that addition of HgCl2 causes the liberation of cuprous ions from MTs followed by a reaction with oxygen, leading to hydroxyl radical formation through a Fenton-type Haber-Weiss reaction.