We have used quantitative DNase I footprinting and UV-melting studies to examine the formation of DNA triplexes in which the third strand thymines have been replaced by 5-propargylamino-dU (U^P). The intramolecular triplex A₆-L-T₆-L-(U^P)₅T (L = two octanediol residues) shows a single UV-melting transition which is >20° higher than that of the parent triplex A₆-L-T₆-L-T₆ at pH 5.5. Although a single transition is observed at all pHs, the melting temperature (Tm) of the modified oligonucleotide decreases at higher pHs, consistent with the requirement for protonation of the amino group. A similar intramolecular triplex with a longer overhanging duplex shows two melting transitions, the lower of which is stabilised by substitution of T by U^P, in a pH dependent fashion. Triplex stability increases by ∼12 K for each T to U^P substitution. Quantitative footprinting studies have examined the interaction of three U^P-containing 9mer oligonucleotides with the different portions of the 17mer sequence 5′-AGGAAGAGAAAAAAGAA. At pH 5.0, the U^P-containing oligonucleotides footprint to much lower concentrations than their T-containing counterparts. In particular (U^P)₆CU^PT binds ∼1000-fold more tightly than the unmodified oligonucleotide T₆CTT. Oligonucleotides containing fewer U^P residues are stabilised to a lesser extent. The affinity of these modified third strands decreases at higher pHs. These results demonstrate that the stability of DNA triplexes can be dramatically increased by using positively charged analogues of thymine.