We have used two-dimensional NMR spectroscopy to study an intramolecular pyrimidine-purine-pyrimidine tripler composed of a Watson-Crick paired DNA duplex and an RNA third strand connected by two hexakis(ethylene glycol) linkers. The solution structure of the tripler was calculated using restrained molecular dynamics and relaxation matrix refinement. The overall helical structure of the Watson-Crick duplex part of the tripler more closely resembles B-DNA than A-DNA. Thus, binding of a third-strand RNA in the major groove does not cause a structural transition toward A-DNA. Both the spectroscopic data and the calculated structures show a narrowing of the minor groove relative to an all-DNA tripler of the same sequence. The sugar puckers of the DNA residues are mostly C2＇-endo while the RNA third strand residues show only partial C3＇-endo character.