Chymotrypsin exists in at least two conformations between pH 2 to 12. One conformation is able, and the other unable, to bind specific substrates and inhibitors. The equilibrium between the two is controlled by a salt bridge between the α-ammonium ion of Ile-16 and the carboxylate ion of Asp-194. Deprotonation of this salt bridge decreases the stability of the active conformation by 2.9 kcal. mole. At 25° C and ionic strength 0.1, 90% of δ-chymotrypsin is in the active conformation between pH 6 and 8. There is a transition, governed by an apparent pK a of 9.08, as the salt bridge is deprotonated, to a high pH equilibrium where only 12% is in the active form. The active fraction also drops at low pH. The pK a of Ile-16 when in the salt bridge is 9.96; but when Ile-16 is not constrained the pK a falls to a normal 7.85. A similar transition is observed in α-chymotrypsin governed by an apparent pK a of 8.76, and in which the pK a of unconstrained Ile-16 is 7.94. The enthalpy of ionization of Ile-16 is 9 kcal. mole when unconstrained. A study at 37° C reveals that, at neutral pH, the transition involving the formation of the salt bridge involves an adverse enthalpy term but is favoured by entropy. The interpretation is based upon knowledge of the enzyme structure derived from X-ray diffraction studies. This affords the first opportunity for measuring the energy of a protein salt bridge.