The hydrophobic component to the binding affinities of one acyclic phosphinate (4) and three macrocyclic phosphonamidate inhibitors (1-3) to the zinc peptidase thermolysin was probed by varying the solvent composition. Increasing the percentage of ethanol in the buffer solution over the range 0-9% increases the inhibition constants, K(i), by up to an order of magnitude. This approach represents an experimental method for distinguishing solvation from conformational or other effects on protein-ligand binding. The size of the "antihydrophobic effect" is correlated with the amount of hydrophobic surface area sequestered from solvent on association of the inhibitor and enzyme, although it is attenuated from that calculated from the surface tension of ethanol-water mixtures. The results are consistent with the Lum-Chandler-Weeks explanation for the size dependence of the hydrophobic effect.