We have studied the effect of the nature of the third-strand sugar (ribose or deoxyribose) on the geometry and stability of triple helices with a pyrimidine motif targeting the polypurine tract of the Friend murine retrovirus. Comparison between triplexes containing a third strand formed by a deoxy 13mer d(TCT5C6), the same oligomer but with C5-methylated cytosines d(T5meCT5(5me)C6), and an analogous modified 13mer RNA 2'Omer(UCU5C6) shows that the sugar conformations of the different triple helices, determined by FTIR spectroscopy, differ depending on nature of the third-strand sugar. Pyrimidine*purine-pyrimidine triple-helix formation with the third-strand RNA and the duplex as DNA appears to be associated with a conversion of the duplex part from a B-form secondary structure with S-type sugars to a geometry in which the polypurine strand sugars adopt an N-type conformation. Thermal dissociation of the triplexes was studied by UV absorbance spectroscopy. The most stable triple helix is obtained when the third strand contains 2'-O-methylated ribose sugars.