A facile synthesis of oligodeoxynucleotides (ODN) containing 2'-deoxy-6-thioinosine (dI6S) based on the convertible nucleoside O6-phenyl-2'-deoxyinosine is presented. After standard solid-phase DNA synthesis and removal of the cyanoethyl protecting groups with DBU treatment with aqueous sodium hydrogen sulfide introduces the sulfur functionality, deprotects the other nucleobases and cleaves the ODN from the solid support in a one-pot reaction. In addition, the extinction coefficient of 2'-deoxy-6-thioinosine is determined by enzymatic fragmentation of the resulting ODN in the presence of adenosine deaminase.