Understanding the structural basis of inhibitor-enzyme interactions, important for the design of new drugs, requires a complete thermodynamic characterization of the binding process as well as a description of the structure of the complex. In this paper, the binding of p-substituted benzamidinium derivatives to the structurally well-characterized serine proteinase bovine pancreatic trypsin has been studied using isothermal titration calorimetry. These experiments have permitted a complete characterization of the temperature dependence of the inhibitor-binding thermodynamics. At 25 degrees C, both the enthalpy and entropy of binding are favourable for all studied derivatives, but this is only true for a relatively narrow temperature range. As binding is characterized by a negative change in heat capacity, the process is characterized by enthalpy--entropy compensation, resulting in a change of the net thermodynamic driving force for association from entropic to enthalpic with increasing temperature. These phenomena are not unusual when hydrophobic forces play an important role. The trend in the relative binding potencies can, to a significant extent, be attributed to the electron-donating/withdrawing character of the substituent at the para position, as shown by the Hammett sigma(p)(+) plot for the different inhibitors; the more polar the p-substituted benzamidine, the less potent it will be as a trypsin inhibitor. This behaviour might result from a bulk solvation effect, meaning that the more polar, lower potency inhibitors will be more stabilized in water than the less polar, higher potency inhibitors.