We have previously shown that the linkage of temperature-dependent protonation and DNA base unstacking equilibria contribute significantly to both the negative enthalpy change (ΔHobs) and the negative heat capacity change (ΔCp,obs) for Escherichia coli SSB homotetramer binding to single-stranded (ss) DNA. Using isothermal titration calorimetry we have now examined ΔHobs over a much wider temperature range (5-60 °C) and as a function of monovalent salt concentration and type for SSB binding to (dT)70 under solution conditions that favor the fully wrapped (SSB)65 complex (monovalent salt concentration ≥0.20 M). Over this wider temperature range we observe a strongly temperature-dependent ΔCp,obs. The ΔHobs decreases as temperature increases from 5 to 35 °C (ΔCp,obs <0) but then increases at higher temperatures up to 60 °C (ΔCp,obs >0). Both salt concentration and anion type have large effects on ΔHobs and ΔCp,obs. These observations can be explained by a model in which SSB protein can undergo a temperature- and salt-dependent conformational transition (below 35 °C), the midpoint of which shifts to higher temperature (above 35 °C) for SSB bound to ssDNA. Anions bind weakly to free SSB, with the preference Br- > Cl - > F-, and these anions are then released upon binding ssDNA, affecting both ΔHobs and ΔCp,obs. We conclude that the experimentally measured values of ΔCp,obs for SSB binding to ssDNA cannot be explained solely on the basis of changes in accessible surface area (ASA) upon complex formation but rather result from a series of temperature-dependent equilibria (ion binding, protonation, and protein conformational changes) that are coupled to the SSB-ssDNA binding equilibrium. This is also likely true for many other protein-nucleic acid interactions.