Endonuclease III (Endo III) is a base excision repair enzyme that recognizes oxidized pyrimidine bases including thymine glycol. This enzyme is a glycosylase/lyase and forms a Schiff base-type intermediate with the substrate after the damaged base is removed. To investigate the mechanism of its substrate recognition by X-ray crystallography, we have synthesized oligonucleotides containing 2'-fluorothymidine glycol, expecting that the electron-withdrawing fluorine atom at the 2' position would stabilize the covalent intermediate, as observed for T4 endonuclease V (Endo V) in our previous study. Oxidation of 5'- and 3'-protected 2'-fluorothymidine with OsO4 produced two isomers of thymine glycol. Their configurations were determined by NMR spectroscopy after protection of the hydroxyl functions. The ratio of (5R,6S) and (5S,6R) isomers was 3:1, whereas this ratio was 6:1 in the case of the unmodified sugar. Both of the thymidine glycol isomers were converted to the corresponding phosphoramidite building blocks and were incorporated into oligonucleotides. When the duplexes containing 2'-fluorinated 5R- or 5S-thymidine glycol were treated with Escherichia coli endo III, no stabilized covalent intermediate was observed regardless of the stereochemistry at C5. The 5S isomer was found to form an enzyme-DNA complex, but the incision was inhibited probably by the fluorine-induced stabilization of the glycosidic bond.