The structural basis for the extraordinary stability of a triple-stranded oligonucleotide in which the third strand contains 2'-aminoethoxy-substituted riboses is investigated by NMR spectroscopy. The enhanced stability of the modified triplex in comparison to the unmodified DNA triplex of the same sequence can be attributed to strong interactions of the aminoethoxy groups of the third strand with the phosphate groups of the purine strand. In molecular dynamics calculations the aminoethoxy side chain was found to be rather flexible, allowing for the presence of hydrogen bonds between the aminoethoxy group of the third strand and two different phosphates of the backbone of the second strand. To investigate the conformational preference of the aminoethoxy side chain a new NMR method has been developed which relies on CH−CH dipolar−dipolar cross-correlated relaxation rates. The results indicate that the aminoethoxy side chains adopt mainly a gauche⁺ conformation, for which only one of the two hydrogen bonds inferred by NMR and molecular dynamics simulations is possible. This demonstrates a highly specific interaction between the amino group of the third strand and one of the phosphate groups of the purine strand.