Formation of natural intramolecular triple-helical structures of DNA is still an intriguing research topic in view of the possible involvement of these structures in biological processes. The biochemical and biophysical properties of DNA triplex structures have been extensively studied, and experimental data show that H-DNA is likely to form in vivo and may regulate the expression of various genes. However, direct and unambiguous evidence of the possible biological roles of these structures is yet elusive. This review focuses on the basic facts that are in favor of, or against, the hypothesis of the presence and function of natural DNA triple-helical structures in vivo, and outlines the different methods and probes that have been used to support these facts.DOI 10.1007/s00018-003-3046-3 - 2003