The solution structure of an intramolecular triple helical oligonucleotide has been solved by NMR. The third strand of the pyrimidine·purine·pyrimidine triplex is composed of 2‘-aminoethoxy-modified riboses, whereas the remaining part of the nucleic acid is DNA. The structure around the aminoethoxy modification was obtained with the help of selective isotope labeling in conjunction with isotope-editing experiments. Dinucleotide steps and interstrand connectivities, as well as the complete backbone conformation of the triplex, were derived from J-couplings, NOEs, and ³¹P chemical shifts. The structure of this triplex, solved by distance geometry, explains the extraordinary stability and increase in rate of triplex formation induced by 2‘-aminoethoxy-modified oligonucleotides:  apart from the formation of seven base triples, a well-defined hydrogen-bonding network is formed across the Crick−Hoogsteen groove involving the amino protons of the aminoethoxy moieties and the phosphates of the purine strand of the DNA. The modified strand adopts a conformation which is close to an A-type helix, whereas the DNA duplex conformation is best described as an unwound B-type helix. The groove dimensions and helical parameters of the 2‘-aminoethoxy-modified rY·dRdY triplex are surprisingly well conserved in comparison with DNA triplexes.