The influence of temperature on the energetics of oligonucleotide-directed triple-helix formation has been investigated in mixed valence salt solutions at pH 7.0. Equilibrium constants for formation of the local pyrimidine.purine.pyrimidine structure afforded by binding of the oligonucleotide 5’-d(T*TTTTCTCTCTCTCT)-3’ to a single 15-bp site within a 339-bp plasmid fragment were measured using quantitative affinity cleavage titrations between 8 and 37 degrees C. In three different solutions buffered by 10 mM Bis-Tris at pH 7.0, BTNS [100 mM NaCl and 1 mM spermine tetrahydrochloride (SpmCl(4))], BTP1 (10 mM NaCl, 140 mM KCl, 1 mM MgCl2, and 1 mM SpmCl(4)), and BTP4 (10 mM NaCl, 140 mM KCl, 1 mM MgCl2, and 4 mM SpmCl(4)), the equilibrium association constants decreased at least 100-fold (from > 10(7) M(-1) to ca. 10(5) M(-1)) as the temperature was increased from 8 to 37 degrees C. Least squares analysis of van’t Hoff plots (In K versus 1/T) of the data revealed that in each solution the tripler is enthalpically stabilized by ca. 2 kcal per mol of base triplets. This average value for single-site tripler formation on large DNA at pH 7.0 in mixed valence salt solutions, which reflects contributions from both T.AT and C+GC base triplets, is consistent with those reported previously for a number of oligonucleotide triplexes in solutions containing single cationic species and in excellent agreement with the calorimetrically determined enthalpy for binding of this 15mer to a 21-bp oligonucleotide DNA duplex.