The heats of reaction of trypsin with soybean (Kunitz), ovomucoid, and lima bean inhibitors have been measured at pH 5.0 and 10° and 25°. All the reactions were endothermic, with ΔH ranging from 8.6 Cal per mole for the lima bean inhibitor to 15.3 Cal per mole for the soybean inhibitor. Equilibrium constants for the association were calculated from the dependence of residual tryptic activity upon added inhibitor. Free energy changes ranged from -9.8 to -12.7 Cal per mole, and ΔS varied from 48 to 79 cal per mole degree. The change in heat capacity, calculated from (ΔH298 - ΔH283)/15, varied from -263 to -442 cal per mole degree. The reactions are in general characterized by large negative ΔG°, moderate positive ΔH, large positive ΔS, and moderately large -ΔCp. The thermodynamic quantities for conversion of native soybean inhibitor to its state in which the arg 64-ile 65 peptide bond is cleaved were calculated as ΔG°298 = -1400 cal per mole, ΔH298 = -4040 cal per mole, ΔS298 = -8.8 cal per mole degree, and ΔCp = -55 cal per mole degree. The results for complex formation and for conversion of native soybean inhibitor to its hydrolyzed form are thought to indicate that interactions other than those directly involved in changes in covalent bonding are thermodynamically significant.