The mechanism of inhibition of the two glucoamylases from a Rhizopus sp. and Aspergillus saitoi by aminoalcohol derivatives was investigated. Hydrolysis of maltose by the glucoamylases was inhibited competitively by aminoalcohols at pH 5.0, and tris(hydroxymethyl)aminomethane, 2-amino-2-ethyl-1,3-propanediol and 2-aminocyclohexanol were relatively good inhibitors of the glucoamylases among the aminoalcohol derivatives tested. One hydroxyl group and an amino group in these inhibitors were indispensable for the inhibitory action, and the addition of other hydroxyl, amino or ethyl groups was enhancing. With an increase in pH from 4.0 to 6.0, the Ki values of the aminoalcohols decreased. This result suggested the participation of a carboxyl group, which was related to the glucoamylase activity and had a pKa of 5.7, in the binding of aminoalcohols. The UV difference spectra induced on binding of the aminoalcohol analogues with the glucoamylases may indicate a change of the environment of tryptophan residues to a slightly higher pH on inhibitor binding. The influence of aminoalcohols on the fluorescence intensity due to tryptophan residues and the CD-spectra of the glucoamylases was less than that of maltitol. Thus, the interaction of aminoalcohols with tryptophan residues in the glucoamylases might be less pronounced than that in the case of substrate analogues. The modes of binding of the aminoalcohols with the two glucoamylases were very similar. Therefore, the phenomenon might be a common feature of glucoamylases in general.