Oligonucleotides uniformly modified with 2'-O,4'-C-ethylene-bridged nucleic acid (ENA) units were synthesized using the phosphoramidite method on a hundred-milligram scale for the evaluation of thermodynamic and chemical properties. The properties of these ENA oligonucleotides with the sequences targeted to human telomerase RNA subunit (hTR) were compared with those of GRN163, which is an oligonucleotide modified with N3'-P5' thiophosphoramidates. The melting temperatures of the duplexes of ENA oligonucleotides with complementary RNA were higher than that of the duplex of GRN163. Moreover, ENA oligonucleotide ENA-13 was more highly stable than GRN163 under acidic conditions (pH 5.0). ENA-13, which contained contiguous guanine sequences, could not form a G-quadruplex, which formation is not feasible for binding to hTR as an antisense molecule. The above findings suggest that ENA oligonucleotides may be useful for antisense therapeutic applications.