The duplex-forming activities of oligonucleotides can be photomodulated by incorporation of an azobenzene unit. Upon isomerizing the trans-azobenzene to the cis form by irradiation with UV light, the T(m) value of the duplex (with the complementary DNA) is lowered so that the duplex is dissociated. The duplex is formed again when the cis-azobenzene is converted to the trans-azobenzene by irradiation with visible light. The photoregulation is successful irrespective of the position of the azobenzene unit in the oligonucleotides. The trans-azobenzene in the oligonucleotides intercalates between two DNA base pairs in the duplexes and stabilizes them because of a favorable enthalpy change. The nonplanar structure of a cis-azobenzene is unfavorable for such an interaction. These photoresponsive oligonucleotides are promising candidates for the regulation of various bioreactions.