Emerging data suggest an important role for cytosine methylation in tumorigenesis. Simultaneously, recent studies indicate a significant contribution of endogenous oxidative DNA damage to the development of human disease. Oxidation of the 5-methyl group of 5-methylcytosine (5mC) residues in DNA results in the formation of 5-(hydroxymethyl)cytosine (hmC). The biological consequences ofhmC residues in vertebrate DNA are as yet unknown; however, conversion of the hydrophobic methyl group to the hydrophilic hydroxymethyl group may substantially alter the interaction of sequence-specific binding proteins with DNA. Central to both biophysical and biochemical studies on the potential consequences of specific DNA damage products such as hmC are efficient methods for the synthesis of oligodeoxynucleotides containing such modified bases at selected positions. In this paper, we describe a method for the placement of hmC residues in oligodeoxynucleotides using established phosphoramidite chemistry. In addition, we have examined the influence of specific hmC residues on enzymatic cleavage of oligodeoxynucleotides by the methylation-sensitive restriction endonucleases MspI and HpaII.