The anticodon domain of lysine transfer ribonucleic acid (tRNA) is a model system for investigation of the structural and biochemical effects of nucleoside posttranscriptional modification. To enable detailed study of the biophysical and structural effects of hypermodified nucleosides, methods have been developed to synthesize RNA oligonucleotides containing the modified nucleosides found in lysine tRNA. We describe in detail the synthesis of protected phosphoramidites of the nucleosides methylaminomethyl-2-thiouridine (mnm5 s2 U), methylcarboxymethyl-2-thiouridine (mcm5 s2 U), and 2-thiomethyl-N -6-carbamoylthreonyl-adenosine (ms2 t6 A). We also describe methods for using these nucleoside phosphoramidite reagents to synthesize RNA oligonucleotides with modified nucleosides incorporated at the specific sequence locations corresponding to their positions in the native lysine tRNAs.