G . U pairs occur frequently and have many important biological functions. The stability of symmetric tandem G . U motifs depends both on the adjacent Watson-Crick base pairs, e.g., 5'G > 5'C, and the sequence of the G . U pairs, i.e., 5'-<(UG)over bar>-3' > 5'-<(GU)over bar>-3', where an underline represents a nucleotide in a G . U pair [Wu, M., McDowell, J. A., and Turner, D. H. (1995) Biochemistry 34, 3204-3211]. In particular, at 37 degrees C, the motif 5'-C<(GU)over bar>G-3' is less stable by approximately 3 kcal/mol compared with other symmetric tandem G . U motifs with G-C as adjacent pairs: 5'-G<(GU)over bar>C-3', 5'-G<(UG)over bar>C-3', and 5'-C<(UG)over bar>G-3'. The solution structures of r(GAG<(UG)over bar>CUC)(2) and r(GGC<(GU)over bar>GCC)(2) duplexes have been determined by NMR and restrained simulated annealing. The global geometry of both duplexes is close to A-form, with some distortions localized in the tandem G . U pair region. The striking discovery is that in r(GGC<(GU)over bar>GCC)(2) each G . U pair apparently has only one hydrogen bond instead of the two expected for a canonical wobble pair. In the one-hydrogen-bond model, the distance between GO6 and UH3 is too far to form a hydrogen bond. In addition, the temperature dependence of the imino proton resonances is also consistent with the different number of hydrogen bonds in the G . U pair. To test the NMR models, U or G in various G . U pairs were individually replaced by N3-methyluridine or isoguanosine, respectively, thus eliminating the possibility of hydrogen bonding between GO6 and UH3. The results of thermal melting studies on duplexes with these substitutions support the NMR models.