The bacterial decoding region of 16S ribosomal RNA has multiple modified nucleotides. In order to study the role of N(4),2'-O-dimethylcytidine (m(4)Cm), the corresponding phosphoramidite was synthesized utilizing 5'-silyl-2'-ACE chemistry. Using solid-phase synthesis, m(4)Cm, 5-methylcytidine (m(5)C), 3-methyluridine (m(3)U), and 2'-O-methylcytidine (Cm) were site-specifically incorporated into small RNAs representing the decoding regions of different bacterial species. Biophysical studies were then used to provide insight into the stabilizing roles of the modified nucleotides. These studies reveal that methylation of cytidine and uridine has different effects. The same modifications at different positions or sequence contexts within similar RNA constructs also have contrasting roles, such as stabilizing or destabilizing the RNA helix.