The dependence on solvent composition of the affinity of thermolysin (E.C. 3.2.24.4) for an immobilized inhibitor was characterized using zonal, competitive and frontal analytical high-performance liquid affinity chromatography (HPLAC). Affinity, measured as the extent of thermolysin retardation on inhibitor (Gly- D-Phe) columns, was strongly affected by variations in the pH, ionic strength, nature of buffering salts and the presence of organic solvents such as 2-propanol in the eluting buffer. While zonal elution experiments allowed the determination of the influence of the mobile phase composition on the overall process, frontal elutions identified specific effects on the individual affinity properties of the enzyme and the immobilized inhibitor. In agreement with data determined previously completely in solution, the interaction on the solid phase was driven mainly by hydrophobic interactions and was affected negatively by organic salts. Based on these results, more efficient elution conditions for the purification of thermolysin by affinity chromatography were established.