O(6)-Methylguanine (O(6)meG) is one of the most toxic, mutagenic, and carcinogenic lesions caused by the interaction of DNA with several catabolism products as well as with environmental methylating agents. Carcinogenic impact of O(6)meG can be conditioned not only by its mutagenic properties but also by alteration in enzymatic methylation of the C5 carbon atom of cytosine residue in CpG sequences. In this study, the effect of O(6)meG on DNA methylation by the catalytic domain of murine DNA methyltransferase (MTase) Dnmt3a (Dnmt3a-CD) is assessed. Damaged DNA duplexes cooperatively bind with Dnmt3a-CD, and O(6)meG changes the stability of enzyme-substrate complexes. Kinetic analysis of the methylation reaction revealed that O(6)meG varies the ratio of productive and nonproductive enzyme-substrate complexes and, depending on localization in substrate, causes decrease or increase in DNA methylation. Dnmt3a-CD is less sensitive to the presence of O(6)meG in DNA substrate than procaryotic MTase SssI recognizing CpG.