EcoRII DNA methyltransferase (M.EcoRII) recognizes the DNA sequence 5´?CC*T/AGG?3´ and catalyzes the transfer of the methyl group from S-adenosyl-L-methionine to the C5 position of the inner cytosine residue (C*). We obtained several DNA duplexes containing photoactive 5-iodo-2´-deoxyuridine (i⁵dU) or 5-[4-(3-(trifluoromethyl)-3H-diazirin-3-yl)phenyl]-2´-deoxyuridine (Tfmdp-dU) to characterize regions of M.EcoRII involved in DNA binding and to investigate the DNA double helix conformational changes that take place during methylation. The efficiencies of methylation, DNA binding affinities and M.EcoRII-DNA photocrosslinking yields strongly depend on the type of modification and its location within the EcoRII recognition site. The data obtained agree with the flipping of the target cytosine out of the DNA double helix for catalysis. To probe regions of M.EcoRII involved in DNA binding, covalent conjugates M.EcoRII-DNA were cleaved by cyanogen bromide followed by analysis of the oligonucleotide-peptides obtained. DNA duplexes containing i⁵dU or Tfmdp-dU at the central position of the recognition site, or instead of the target cytosine were crosslinked to the Gly²⁶⁸-Met³⁹¹ region of the EcoRII methylase. Amino acid residues from this region may take part both in substrate recognition and stabilization of the extrahelical target cytosine residue.