Formation and dissociation of DNA triplex are reversibly photoregulated by cis <----> trans isomerization of the azobenzene tethered to the third strand. When the azobenzene takes the trans from, a stable triplex is formed. Upon the isomerization of trans-azobenzene to its cis form by UV light irradiation (300 < lambda < 400 nm), however, the modified oligonucleotide is removed from the target duplex. The triplex is re-formed on photoinduced cis <----> trans isomerization (lambda > 400 nm). The photoregulating activity significantly depends on the position of azobenzene in the third strand, as well as on the geometric position (meta or para) of its amido substituent. For m-amidoazobenzene, the photoregulation is the most effective when it is tethered to the 5'-end of the third strand. However, p-amidoazobenzene should be introduced into the middle of the strand for effective regulation. In the optimal cases, the change of T-m of the triplex, caused by the cis <----> trans isomerization of azobenzene, is greater than 30 degreesC. UV-visible and CID spectroscopy, as well as computer modeling studies, clearly demonstrate that the trans-azobenzene intercalates between the base pairs in the target duplex and thus stabilizes the triplex by stacking interactions. On the other hand, nonplanar cis-azobenzene destabilizes the triplex due to its steric hindrance against the adjacent base pairs.