Attempts to incorporate the phosphoramidite of the thymine-thymine (6-4) photoproduct C5 thiol analogue (s(5)T(6-4)T PP), whose sulfur atom was protected with the methylsulfenyl group, into oligodeoxynucleotides (ODNs), are reported. Using matrix-assisted laser desorption-ionisation mass spectrometry (MALDI-MS) coupled to enzymatic digestion, accurate mass measurements and tandem mass spectrometry experiments, we demonstrated that ODNs containing the (2-cyanoethylthio)(5)T(6-4)T PP were obtained. Supported by model reactions, these results were explained 1) by the incorporation, during oligonucleotide synthesis, of the sulfur deprotected phosphoramidite that arose from a Michaelis-Arbusov-type rearrangement, and 2) the Michael addition to the thiol of acrylonitrile released upon the cyanoethyl phosphotriester deprotection. To avoid the formation of the cyanoethyl adduct, the phosphotriester deprotection was carried out in the presence of a thiol in excess. This afforded the ODN containing the h(5)T(6-4)T PP.