In order to monitor DNA flexibility, we have recently reported the design of an artificial DNA bending system consisting of two triple helix forming oligonucleotides (TFOs) connected by a flexible linker [Akiyama, T., & Hogan, M. E. (1996) Proc. Natl. Acad. Sci. U.S.A. 93, 12122-12127], which spans a single turn of DNA helix. Those data suggested that up to 60 degrees of bending deformation could be induced with an expenditure of energy which is much smaller than predicted from bulk flexibility parameters. In this report, the detailed structure of the bend has been investigated by three different methods: circular permutation analysis, phasing analysis, and ring closure. Circular permutation and phasing analysis suggest that the magnitude of the bend is dependent on linker length. The apparent location of the bend was estimated from circular permutation analysis to be at the duplex region intervening the two sites of triple helix formation. The electrophoretic mobility of the bent complex appears to vary with the sequence of the intervening duplex region of the binding site complex, in the order of AT-rich > random > or = GC-rich sequence. Detailed fitting of the phasing data has shown that bending is not accompanied by significant twisting deformation. Ring closure analysis with T4 DNA ligase has confirmed the general magnitude of the TFO-induced bend and has additionally suggested that formation of the simple linear antiparallel triple helix does not enhance DNA flexibility.