The thymidine stereoisotopomer, (2’S)-[2’-H-2]thymidine, which incorporates deuterium in the S configuration at the furanosyl 2’ carbon, has been synthesized and its vibrational spectra have been recorded and compared with those of normal thymidine. Infrared and Raman spectra were collected from crystalline powders, the latter using 1063-, and 514.5-nm excitations; ultraviolet resonance-Raman spectra were collected from aqueous solutions using 244-nm excitation. The results show, remarkably, that virtually all normal modes of thymidine involve some degree of vibrational coupling between the thymine base residue and the deoxyribose moiety. Nevertheless, systematic assignments and correlation of the spectral frequencies of thymidine and (2’S)-[2’-H-2]thymidine have been accomplished. A finding of importance for nucleic acid structure applications is that many prominent Raman marker bands of thymidine, assigned previously as thymine ring vibrations, in fact involve appreciable coupling with the C2’ methylene group of the attached sugar. Vibrational coupling between the base and sugar groups implies frequency dependence upon sugar conformation and allows the bands in question to be exploited as markers of deoxyribose ring pucker and glycosyl orientation in Raman spectra of DNA, antiviral drugs, and other thymine-containing nucleoside analogues. The present results also enable unambiguous and novel assignment of spectral bands to specific vibrational modes of the C2’ methylene group of thymidine as follows : C2’H-2 antisymmetric stretching (2995 cm(-1)), symmetric stretching (2956 cm(-1)), scissoring (1404 cm(-1)), and wagging (1174 cm(-1)). Additionally, probable assignments are deduced for the C2’H-2 twisting (1103 cm(-1)) and-rocking modes (898 cm(-1)). Normal mode assignments are also proposed for many other vibrational bands of thymidine.