The preparation of a pentadecanucleoside tetradecaphosphate containing the modified base 5,6-dihydrothymine (DHT) is reported herein. The synthesis was performed with a mixture of diastereoisomers (5R and 5S), obtained by catalytic hydrogenation of thymidine. The phosphorylated protected monomer was characterized by proton NMR and FAB mass spectrometry. It was introduced into the DNA fragment (poly dT) by the solid phase phosphotriester approach. After deprotection, the determination of the site of the modified base in the chain was made using different methods. The classical chemical sequencing method of Maxam and Gilbert showed no difference with respect to thymine moieties in the pentadecamer. To characterize the presence and the location of the modified base DHT, the 32P 5'-end labelled pentadecamer was heated in formic acid at 90 degrees C and length separated by polyacrylamide gel electrophoresis. The Electronic Impact mass spectrometry under pyrolytic conditions of the 15-mer showed that the modified base was present in the deprotected DNA fragment.