The effect of water activity on the rate of thermolysin-catalyzed synthesis of an aspartame precursor has been investigated in water-miscible and water-immiscible solvents. In both cases, the enzyme reaction rate at a given water activity was found to be significantly different depending on the nature of the solvent. The reaction rates in water-immiscible solvents, where the water activities were close to 1.0, were found to be significantly dependent on the volume ratio of water to organic media and the hydrophobicity of the solvent. These data suggest that the enzyme reaction in the solvent is influenced appreciably by other factors in addition to the water activity.