We have developed a new method for the preparation of oligodeoxyribonucleotides and oligo(2'-O-methylribonucleotides) that contain a 2'-phosphorylated ribonucleoside residue, and optimized it to avoid 2' -3' -isomerization and chain cleavage. Structures of the 2' -phosphorylated oligonucleotides were confirmed by MALDI-TOF MS and enzymatic digestion, and the stability of their duplexes with DNA and RNA was investigated. 2'-Phosphorylated oligonucleotides may be useful intermediates for the introduction of various chemical groups for a wide range of applications.