Locked nucleic acids (LNA) considerably enhance the thermodynamic stability of DNA and RNA duplexes. We report the thermodynamic stabilities of LNA-2'-O-methyl RNA/RNA duplexes designed to provide insight into the contributions of stacking and hydrogen bonding interactions to the enhanced stability. The results show that hydrogen bonding of LNA nucleotides is similar to that of 2'-O-methyl RNA nucleotides, whereas the 3'-stacking interactions are on average approximately 0.7 kcal/mol more favorable at 37 degrees C than for 2'-O-methyl or RNA nucleotides. Moreover, NMR spectra suggest helical preorganization of the single-stranded tetramer, C(L)A(M)A(L)U(M), probably due to restriction of some torsion angles. Thus, enhanced stacking interactions and helical preorganization of single-stranded oligonucleotides contribute to the extraordinary stabilization of duplexes by LNA nucleotides.