Interactions of oligonucleotides comprising (1-beta-D-2'-deoxy-threo-pentafuranosyl)thymine and (1-beta-D-2'-deoxy-threo-pentafuranosyl)cytosine residues (oligodeoxyxylonucleotides or OXNs) with complementary single-stranded DNA fragments were investigated. Using nondenaturing gel electrophoresis, footprinting, and melting assays, pyrimidine OXNs were shown to form triplexes with the purine DNA template, which are stable at neutral pH and comparable in heat stability with the corresponding natural polypurine-polypyrimidine DNA duplexes. In such triplexes, the N3 of cytosines in one of the OXNs are protonated. As revealed by CD spectroscopy in the 210-340 nm range, the form of the triple helix depends on the nucleotide composition and sequence of the DNA template, and is intermediate between A and B.