Studies of a series of short oligonucleotide double and triple helices containing either all RNA, all DNA, or a mixture of the two show strand-dependent variation in their stability and structure. The variation in stability for both groups falls over a range of greater than 10 kilocalories per mole. In forming the triple helix, RNA is favored on both pyrimidine strands, whereas DNA is favored on the purine strand. In general, relatively unstable duplexes form particularly stable triplexes and vice versa. Structural data indicate that the strands in hybrid helices adopt a conformation that is intermediate between molecules containing all DNA and all RNA. Thus, RNA-DNA hybrids were not forced into the conformation of the RNA (A-form). The provocative stability of the triplex with an RNA third strand+DNA duplex points to novel antisense strategies and opens the possibility of an in vivo role of these structures. Overall, the data emphasize the fundamental role of sugars in determining the properties of nucleic acid complexes.