The conformational characteristics ofN4-acetyl-2′-O-methylcytidine (ac4Cm), which is one of the modified cytidines unique to the tRNA of extremely thermophilic archaebacteria, and related nucleosides,N4-acetylcytidine (ac4C), 2′-O-methylcytidine (Cm) and cytidine, were analyzed by proton nuclear magnetic resonance spectroscopy. Ribose methylation andN4-acylation were found to confer high conformational rigidity to the ribose moiety, suggesting that these post-transcriptional modifications play a role in structural stabilization of tRNA, which is of particular importance at high temperature.