This study investigates the pro-oxidant activity of 3'- and 4'-O-methylquercetin, two relevant phase II metabolites of quercetin without a functional catechol moiety, which is generally thought to be important for the pro-oxidant activity of quercetin. Oxidation of 3'- and 4'-O-methylquercetin with horseradish peroxidase in the presence of glutathione yielded two major metabolites for each compound, identified as the 6- and 8-glutathionyl conjugates of 3'- and 4'-O-methylquercetin. Thus, catechol-O-methylation of quercetin does not eliminate its pro-oxidant chemistry. Furthermore, the formation of these A-ring glutathione conjugates of 3'- and 4'-O-methylquercetin indicates that quercetin o-quinone may not be an intermediate in the formation of covalent quercetin adducts with glutathione, protein and/or DNA. In additional studies, it was demonstrated that covalent DNA adduct formation by a mixture of [4-(14)C]-3'- and 4'-O-methylquercetin in HepG2 cells amounted to only 42% of the level of covalent adducts formed by a similar amount of [4-(14)C]-quercetin. Altogether, these results reveal the effect of methylation of the catechol moiety of quercetin on its pro-oxidant behavior. Methylation of quercetin does not eliminate but considerably attenuates the cellular implications of the pro-oxidant activity of quercetin, which might add to the mechanisms underlying the apparent lack of in vivo carcinogenicity of this genotoxic compound. The paper also presents a new mechanism for the pro-oxidant chemistry of quercetin, eliminating the requirement for formation of an o-quinone, and explaining why methylation of the catechol moiety does not fully abolish formation of reactive DNA binding metabolites.