Isothermal titration calorimetry (ITC) is used to study the thermodynamics of assembly of the three DNA oligonucleotides S1 (5'-GCCTGCCACCGC), S2 (5'-GCGGTGCGTCCG), and S3AA (5'-CGGACGAAGCAGGC) to form a three-way junction (TWJ) complex consisting of three double-helical arms radiating from a junction region having two unpaired adenosines in one strand (S3AA). The thermodynamics of assembly were measured for three different orders of addition of the component oligonucleotides at four temperatures between 10 and 25 degrees C. At each temperature studied, the overall values of delta H, delta S degrees, and delta G degrees for assembly of the complex from the component single strands were found to be independent of the order of addition. The enthalpy of binding, delta H, was found to be linearly dependent on temperature. From the temperature dependence of delta H, the change in heat capacity delta Cp, for the overall assembly of three strands to form the junction complex was calculated and found to be -1.6 kcal mol-1K-1. This work represents the first attempt to evaluate the thermodynamics of DNA TWJ formation by ITC.