We report a direct determination of the thermodynamic contribution that DNA single-stranded order makes to DNA duplex formation. By using differential scanning calorimetry (DSC) and temperature-dependent UV absorbance spectroscopy, we have characterized thermodynamically the thermally induced disruption of the 13-mer duplex [d(CGCATGAGTACGC)].[d(GCGTACTCATGCG)] (henceforth called S1.S2) and its component single strands, [d(CGCATGAGTACGC)] (henceforth called S1) and [d(GCGTACTCATGCG)] (henceforth called S2). These spectroscopic and calorimetric measurements yield the following thermodynamic profiles at 25 degrees C: delta G degree = 20.0 kcal/mol, delta H degree = 117.0 kcal/mol, and delta S degree = 325.4 cal.degree-1.mol-1 for duplex melting of S1.S2; delta G degree = 0.45 kcal/mol, delta H degrees = 29.1 kcal/mol, and delta S degree = 96.1 cal.degree-1.mol-1 for single-strand melting of S1; delta G degree = 1.44 kcal/mol, delta H degree = 27.2 kcal/mol, and delta S degree = 86.4 cal.degree-1.mol-1 for single-strand melting of S2 (1 cal = 4.184 J). These data reveal that the two single-stranded structures S1 and S2 are only marginally stable at 25 degrees C, despite exhibiting rather substantial transition enthalpies. This behavior results from enthalpy and entropy contributions of similar magnitudes that compensate each other, thereby giving rise to relatively small free energies of stabilization for the single strands at 25 degrees C. By contrast, the S1.S2 duplex state is very stable at 25 degrees C since the favorable transition entropy associated with duplex disruption (325.4 cal.degree-1.mol-1) is more than compensated for by the extremely large duplex transition enthalpy (117.0 kcal/mol). We also measured directly an enthalpy change (delta H degree) of -56.4 kcal/mol for duplex formation at 25 degrees C using isothermal batch-mixing calorimetry. This duplex formation enthalpy of -56.4 kcal/mol at 25 degrees C is very different in magnitude from the duplex disruption enthalpy of 117.0 kcal/mol measured at 74 degrees C by DSC. Since the DSC measurement reveals the net transition heat capacity change to be close to zero, we interpret this large disparity between the enthalpies of duplex disruption and duplex formation as reflecting differences in the single-stranded structures at 25 degrees C (the initial states in the isothermal mixing experiment) and the single-stranded structures at approximately 80 degrees C (the final states in the DSC experiment). In fact, the enthalpy for duplex formation at 25 degree C (-56.4 kcal/mol) can be combined with the sum of the integral enthalpies requires to melt each single strand from 25 to 80 degree C (23.6 kcal/mol for S1 and 27.2 kcal/mol for S2) to calculate a delta H degree of -107.2 kcal/mol for the hypothetical process of duplex formation from random-coil unstacked single strands at 25 degree C. The magnitude of this predicted delta H degree value for duplex formation is in good agreement with the corresponding parameter we measure directly by DSC for duplex disruption (117.0 kcal/mol), thereby lending credence to our interpretation and analysis of the data. Thus, our results demonstrate that despite being only marginally stable at 25 degree C, single strands can exhibit intramolecular interactions that enthalpically poise them for duplex formation. For the duplex studied herein, prior to association at 25 degree C, the two complementary single strands already possess > 40% of the total enthalpy (50.8/117) that ultimately stabilizes the final duplex state. This feature of single-stranded structure near room temperature can reduce significantly the enthalpic driving force one might predict for duplex formation from nearest-neighbor data, since such data generally are derived from measurements in which the single strands are in their random-coil states. Consequently, potential contributions from single-stranded structure must be recognized and accounted for when designing hybridization experiments and when using isothermal titration and/or batch mixing techniques to study the formation of duplexes and higher-order DNA structures (e.g., triplexes, tetraplexes, etc.) from their component single strands.