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6-Thioguanine is a cytotoxic drug for the treatment of acute leukaemias but its mode of action is controversial. In vivo 6-thioguanine forms 2’-deoxy-6-thio-guanosine triphosphate which incorporates into DNA. DNA methylation is an epigenetic modification of the genomes of diverse organisms that plays an important role in regulation of many biologic processes including gene regulation. Murine DNA methyltransferase Dnmt3a is involved in establishment of new methylation patterns in genome. The enzyme transfers methyl groups from S-adenosyl-L-methionine to the C-5 position of cytosine at CpG sites. The active form of Dnmt3a is a tetramer with two catalytic centers. We postulate that the incorporation of 6-thioguanine into CpG sites instead of guanine may perturb the Dnmt3a-mediated cytosine methylation at these sites. To test this hypothesis we assessed the impact of 6-thioguanine in CpG on cytosine methylation mediated by catalytic domain of Dnmt3a (Dnmt3a-CD). Dnmt3a-CD retains catalytic activity in the absence of an N-terminal regulatory domain of the enzyme. 30-mer DNA substrate (I) containing two hemimethylated CpG sites separated by 10 base pairs (one helix turn) was designed. 6-thioguanine was introduced into one (substrate II) or both (substrate III) CpG sites instead of guanine. The initial rates of methylation (V0) of DNA substrates I-III by Dnmt3a-CD in 1.7-fold excess of DNA relative to enzyme were measured. The replacement of the CpG guanine by 6-thioguanine both in one or two sites (substrates II and III respectively) led to decrease of the V0 values compared to the parent substrate I. It is consistent with our previous findings about crucial importance of the contact of the 6-oxo group of the CpG guanine residue with Dnmt3a-CD. We found that incorporation of one 6-thioguanine residue (substrate II) caused approximately 2.25-fold decrease of the V0 value whereas additional incorporation of 6-thioguanine residue into the second site (substrate III) had less dramatic effect. One of the main steps in the methylation process is flipping of the target cytosine out of the double helix for its methylation. Absorbtion band of 6-thioguanine in the 300-400nm area (where unmodified DNA is transparent) allowed us to use 6-thioguanine residue as spectroscopic probe to investigate whether 6-thioguanine incorporation into the CpG site affects flipping of the adjacent target cytosine by Dnmt3a-CD. For this purpose we utilized low energy circular dichroism spectroscopy (CD). Addition of Dnmt3a-CD to substrate III slightly changed the shape of substrate III CD spectrum in the 300-400nm area. This fact correlates with the substrate III decreased level of methylation . Taken together, these results showed that 6-thioguanine, after being incorporated into DNA, may perturb the epigenetic pathway of gene regulation.