(L)-alpha-Threofuranosyl-(3'-->2')-oligonucleotides (TNA) containing vicinally connected phosphodiester linkages undergo informational base pairing in an antiparallel strand orientation and are capable of cross-pairing with RNA and DNA. TNA is derived from a sugar containing only four carbon atoms and is one of the simplest potentially natural nucleic acid alternatives investigated thus far in the context of a chemical etiology of nucleic acid structure. Compared to DNA and RNA that contain six covalent bonds per repeating nucleotide unit, TNA contains only five. We have determined the atomic-resolution crystal structure of the B-form DNA duplex [d(CGCGAA)T*d(TCGCG)](2) containing a single (L)-alpha-threofuranosyl thymine (T*) per strand. In the modified duplex base stacking interactions are practically unchanged relative to the reference DNA structure. The orientations of the backbone at the TNA incorporation sites are slightly altered in order to accommodate fewer atoms and covalent bonds. The conformation of the threose is C4'-exo with the 2'- and 3'-substituents assuming quasi-diaxial orientation.