© 2020 American Chemical Society. DNase I footprints of intermolecular DNA triplexes are often accompanied by enhanced cleavage at the 3′-end of the target site at the triplex-duplex junction. We have systematically studied the sequence dependence of this effect by examining oligonucleotide binding to sites flanked by each base in turn. For complexes with a terminal T.AT triplet, the greatest enhancement is seen with ApC, followed by ApG and ApT, with the weakest enhancement at ApA. Similar DNase I enhancements were observed for a triplex with a terminal C+.GC triplet, though with little difference between the different GpN sites. Enhanced reactivity to diethylpyrocarbonate was observed at As that flank the triplex-duplex junction at AAA or AAC but not AAG or AAT. Fluorescence melting experiments demonstrated that the flanking base affected the stability with a 4 °C difference in Tm between a flanking C and G. Sequences that produced the strongest enhancement correlated with those having the lower thermal stability. These results are interpreted in terms of oligonucleotide-induced changes in DNA structure and/or flexibility.