Isothermal titration calorimetry (ITC) was used to test the hypothesis that the relatively small enthalpy change (ΔH(obs)) and large negative heat capacity change (ΔC(p,obs)) observed for the binding of the Escherichia coli SSB protein to single-stranded (ss) oligodeoxyadenylates result from the temperature-dependent adenine base unstacking equilibrium that is thermodynamically coupled to binding. We have determined ΔH(l,obs) for the binding of 1 mole of each of dT(pT)34, dC(pC)34, and dA(pA)34 to the SSB tetramer (20 mM NaCl at pH 8.1). For dT(pT)34 and dC(pC)34, we found large, negative values for ΔH(l,obs) of -75 ± 1 and -85 ± 2 kcal/mol at 25 °C, with ΔC(p,obs) values of -540 π 20 and -570 ± 30 cal mol-1 K-1 (7-50 °C), respectively. However, for SSB-dA(pA)34 binding, ΔH(l,obs) is considerably less negative (-14 ± 1 kcal/mol at 25 °C), even becoming positive at temperatures below 13 °C, and ΔC(p,obs) is nearly twice as large in magnitude (-1180 ± 40 cal mol-1 K-1). These very different thermodynamic properties for SSB-dA(pA)34 binding appear to result from the fact that the bases in dA(pA)34 are more stacked at any temperature than are the bases in dC(pC)34 or dT(pT)34 and that the bases become unstacked within the SSB-ssDNA complexes. Therefore, the ΔC(p,obs) for SSB-ssDNA binding has multiple contributions, a major one being the coupling to binding of a temperature-dependent conformational change in the ssDNA, although SSB binding to unstacked ssDNA still has an 'intrinsic' negative ΔC(p,0). In general, such temperature-dependent changes in the conformational 'end states' of interacting macromolecules can contribute significantly to both ΔC(p,obs) and ΔH(obs).