We have examined the linkage between salt concentration and temperature for the equilibrium binding of the tetrameric Escherichia coli single-stranded binding (SSB) protein to three single-stranded nucleic acids, poly(U), dA(pA)69, and dT(pT)69, by van't Hoff analysis and isothermal titration calorimetry (ITC). For SSB binding to poly(U) in its (SSB)65 mode, the equilibrium association constant, Kobs, decreases with increasing salt concentration at all temperatures examined, and binding is enthalpy-driven; however, the value of ∂ log Kobs/∂ log [NaCl] is highly temperature-dependent, varying from -9.3 ± 03 at 10°C to -5.1 ± 0.4 at 37°C. This indicates that ΔHobs for SSB-poly(U) binding is strongly dependent on [NaCl]: based on van't Hoff analyses, ΔHobs varies from -57 ± 3 kcal/mol at 0.18 M NaCl to -34 ± 3 kcal/mol at 0.42 M NaCl (∂ΔHobs/∂ log [NaCl] = 60 ± 5 kcal/mol). However, ∂ΔHobs/∂ log [NaF] is independent of temperature (25-37°C), indicating that the effect of [NaCl] on ΔHobs is due primarily to Cl-. Similar effects were also observed for SSB binding to dA(pA)69. We also measured ΔHobs and its dependence on [NaCl] for SSB binding to dT(pT)69 by ITC and find ΔHobs = -144 ± 4 kcal/mol (0.175 M NaCl, pH 8.1, 25°C) and ∂ΔHobs/∂ log [NaCl] = 46 ± 2 kcal/mol (0.175-2.0 M NaCl). These large effects of [NaCl] on ΔHobs appear to result, at least partly, from the release of preferentially bound Cl- from SSB protein upon binding nucleic acid, with the release of Cl- being linked to a process with ΔH ≫ 0. Effects of salt concentration on ΔHobs are not observed for processes in which only monovalent cations are released from the nucleic acid, presumably since Na+ or K+ are bound to linear nucleic acids as delocalized, fully hydrated cations. Such salt effects on ΔHobs may serve as a signature for differential ion-protein binding. These results underscore the need to examine the linkage of [salt] to ΔHobs, as well as ΔG°obs and ΔS°obs, in order to understand the bases for stability and specificity of protein-nucleic acid interactions.