This study investigates the role of cellular tyrosinase and/or peroxidase-like oxidative enzyme activity in the covalent binding of quercetin to glutathione, protein, and DNA, as well as the stability of quercetin DNA adducts in time. This was done by studying the formation of glutathionyl quercetin adducts in various in vitro models, and the covalent binding of radiolabeled quercetin to protein and DNA in cells with elevated peroxidase or tyrosinase levels and in cells devoid of nucleotide excision repair (NER). Cells with elevated tyrosinase or peroxidase levels contained approximately 2 times higher levels of covalent quercetin adducts than cells without detectable levels of these oxidative enzymes. However, this difference was smaller than expected based on the differences in tyrosinase and/or peroxidase levels, indicating that these types of oxidative enzyme activities do not play a major role in the cellular prooxidant activity of quercetin. Furthermore, quercetin DNA adducts were of transient nature, independent of the presence of NER, suggesting chemical instability of the adducts. Whether this transient nature reflects real reversibility or formation of genotoxic, depurinated sites remains to be investigated at the molecular level. Together, these data indicate that formation of covalent quercetin adducts can be expected in all cells, independent of their oxidative enzyme levels, whereas the transient nature of the DNA adducts formed may limit or cause their ultimate biological impact. If the transient nature represents chemical reversibility of the adduct formation, it would provide a possible explanation for the apparent lack of in vivo carcinogenicity of this in vitro mutagen. Therefore, in vitro mutagenicity studies should focus more on the transient nature of DNA adducts responsible for the mutagenicity in vitro, since this transient nature of DNA adducts may play an essential role in whether the genotoxicity observed in vitro will have any impact in vivo. © 2005 American Chemical Society. Chemicals / CAS: DNA, 9007-49-2; glutathione, 70-18-8; monophenol monooxygenase, 9002-10-2; oxidoreductase, 9035-73-8, 9035-82-9, 9037-80-3, 9055-15-6; peroxidase, 9003-99-0; protein, 67254-75-5; quercetin, 117-39-5; DNA Adducts; Glutathione, 70-18-8; Monophenol Monooxygenase, EC 1.14.18.1; Peroxidase, EC 1.11.1.7; Proteins; Quercetin, 117-39-5