AbstractHydrolyses of maltose, maltotriose, and soluble starch catalyzed by glucoamylase (Asp. Niger) were carried out in the aqueous solutions of methanol, ethanol, ethylene glycol, and 1,4‐dioxane at 40°C and at the optimum pH in the respective solutions. By the kinetic analysis based on the subsite model, it was shown that the intrinsic rate constant, Kint, was electrostatically affected by the dielectric constant of the hydroorganic solutions. The affinity of the third subsite, A3, which affects the apparent rate constant, K0, was correlated with the ×Gtr's of maltose and amino acid side chains.