This study attempted to comprehend how temperature affects hydrophobic interaction between proteins and hydrophobic adsorbents. By equilibrium batch analysis, we measured the adsorption isotherm to evaluate the protein-adsorbent affinity, while isothermal titration calorimetry was used to measure the adsorption enthalpy. In addition, the affinity and enthalpy differences between two proteins, α-chymotrypsinogen A and trypsinogen, with two adsorbents, butyl-Sepharose and octyl-Sepharose gel, under varying temperatures were studied with respect to the exposed hydrophobic segments of the protein and ligand hydrophobicity. The enthalpies obtained in this investigation can be used to more thoroughly understand the hydrophobic interaction between proteins and adsorbents. First, the adsorption isotherm experiments reveal that the adsorption quantity of the proteins with the Sepharose gels increases with temperature. For a microcalorimetric measurement, as temperature is increased from 298 to 310 K, the ΔH value of α-chymotrypsinogen A with butyl-Sepharose increases, while the ΔH value of trypsinogen is reduced. This is likely due to the fact that α-chymotrypsinogen A has a higher area of exposed hydrophobic segments than trypsinogen does. This observation also implies that as temperature increases, the interaction mechanism of α-chymotrypsinogen A with butyl-Sepharose changes from an adsorption-dominated process to a partitioning process. In addition, for octyl-Sepharose, the ΔH value of α-chymotrypsinogen A is positive and decreases with temperature increment. However, the ΔH value of trypsinogen was positive and increased with temperature. Therefore, we conclude that as temperature increases, the interaction mechanism of the proteins for octyl-Sepharose is a partitioning-dominated process. (C) 2000 Academic Press.