Only a limited number of tools are available to study cytosine methylation in DNA. One of the representatives of the recently discovered methyl-dependent DNA endonucleases is an enzyme GlaI. It is of great interest for determining the methylation status of eukaryotic genomic DNA due to its ability to cleave only methylated DNA. However, the ability of the GlaI endonuclease to recognize oxidized derivatives of 5-methylcytosine (mC), in particular another epigenetic base, 5-hydroxymethylcytosine (hmC), has not yet been characterized. It is not possible to fully use the potential of GlaI in the analysis of methylation due to the notable occurrence of the latter in the DNA of mammals. In this study, the efficiency of cleavage of DNA substrates with various combinations of mC and hmC by methyl-dependent DNA-endonuclease GlaI was compared; the kinetic parameters of cleavage reactions for fully methylated and fully hydroxymethylated recognition site were determined. It was shown that in most cases GlaI recognized substrates containing mC better than substrates containing hmC in the same positions. The most effective hydrolysis of substrates containing modifications in the sequence 5'-GCGC-3'/3'-CGCG-5' required the presence of hmC not only in the central but also in the edge positions in both DNA chains as in the case of mC.