Salt-activation of hydrolytic enzymes in organic solvents has been studied extensively, and partially optimized (e.g., with respect to water content, pH, lyophilization time, salt content, Jones-Dole B coefficient, etc.) to yield near aqueous levels of activity. However, little is understood about the mechanistic details of salt-activation, although increased enzyme flexibility and active site polarity have been hypothesized.  With the aim of elucidating the degree to which salt-activation is an enthalpic and/or entropic effect, variable temperature experiments were performed on subtilisin Carlsberg (SC) co-lyophilized with several inorganic salts.  The results indicate that salt activation induces a greater degree of transition state flexibility, reflected by a more positive entropy of activation, ΔS *, for the correspondingly more active salt preparation (higher (k cat /K m ) app ) in both hexane and acetone.   That the enthalpy of activation, ΔH *, is relatively constant regardless of salt type or salt content indicates preservation of the SC transesterification mechanism.  Furthermore, a correlation between , ΔS *, and a “global” NMR spin lattice relaxation time, T1 ρH , indicative of proton motion on the ms time scale on the enzyme, was observed for the salt preparations studied. These results suggest that greater enzyme flexibility in the more active salt preparations may be responsible for the observed differences in activity.