In order to investigate the duplex structure of DNA and RNA containing an L-nucleotide residue, we carried out the UV-melting and CD experiments. Although the introduction of an L-nucleotide into DNA/DNA, DNA/RNA and RNA/RNA duplexes reduced their Tm values, the typical two-state transitions were observed for all of the duplexes. The incorporation of an L-nucleotide into the RNA strand caused more remarkable decreases of the Tm value than that into the DNA strand. The CD spectra of the DNA/DNA and RNA/RNA duplexes containing an L-nucleotide are similar to those of unmodified duplexes. On the other hand, the spectra of the heterochiral DNA/RNA hybrid duplexes are significantly different from those of the homochiral DNA/RNA hybrid duplexes, which are generally thought to adopt the A-form.