Recognition of B-DNA by oligonucleotides that form triple helices is a unique method to specifically recognize sequences of double-stranded DNA. Recently, some significant limitations of the triple-based applications have been overcome. Stable intermolecular triplexes can be formed under physiologic conditions. Binding affinities of modified oligonucleotides to their target sequence due to Hoogsteen or reverse Hoogsteen hydrogen bonding interactions are now in the range of those obtained for duplex formation via Watson-Crick hydrogen bonding interactions even if the kinetics may be quite different. Progress has been made toward developing general procedures to determine the molecular mechanisms of action of triplex-forming oligonucleotides (TFO) administered to cultured cells to provide a rational proof-of-concept for antigene strategies. The antigene strategy has reached a point where TFOs can be used to interfere with several biologic progresses (replication, transcription, recombination, repair) in relevant systems both in vitro and ex vivo.