A method is described for evaluating the temperature-invariant enthalpy, Delta H degrees(T-0), for chymotrypsinogen A and ribonuclease A in an aqueous glycerol solution in the standard state, to determine the effect of glycerol on the thermodynamic stability of these two proteins. Chymotrypsinogen A has a temperature-invariant enthalpy of 210 kcal mol(-1) at low pH in the absence of glycerol. In 10% aqueous glycerol solution, Delta H degrees(T-0) is 239 kcal mol(-1) in 40% glycerol, this value is 249 kcal mol(-1). The temperature-invariant enthalpy of alpha-chymotrypsin dimerization is only 33 kcal mol(-1), while that required for the conformational transition of ribonuclease A at low pH is approximately 60 kcal mol(-19-13). Solvents such as glycerol tend to lower the melting temperature, [T-m], in chymotrypsinogen A at low pH, possibly due to preferential interaction as a result of solvent ordering in the associated state, rather than a conformational thermal transition from the native to denatured state. Using the Planck-Benzinger thermal work function, it is possible to determine the different types of thermodynamic compensation taking place in these systems, that is, cases in which there is (i) thermodynamic compensation dominated by the temperature-invariant enthalpy, Delta H degrees(T-0); (ii) balanced thermodynamic compensation between Delta H degrees(T-0) and the heat integrals; (iii) thermodynamic compensation among Delta H degrees(T-0), the heat integrals, and Delta AS degrees(T), typical of biological systems; and (iv) thermodynamic compensation dominated by T Delta S degrees(T), the bound energy change, and the heat integrals.