1H-NMR analyses have been made on the conformations of 2-thioribothymidine (s 2 T), 2-thiodeoxyribothymidine (s 2 dT), as well as ribothymidine (T) and deoxyribothymidine (dT). s 2 T and s 2 dT exclusively take the anti form rather than the syn form. The C3'-endo-gg form of the sugar moiety is remarkably stabilized on modification of T to s 2 T, but not on modification of dT to s 2 dT. The steric effects of the 2-thiocarbonyl group and the 2'-hydroxyl group cause the rigidity of the C3'-endo-gg form of s 2 T. Such rigidity of s 2 T probably contributes to the thermostability of 2-thiopyrimidine polyribonucleotides and extreme thermophile tRNAs.