1,5-Anhydro-D-glucitol (1,5-AG) was found to inhibit trehalase and trehalose phosphorylase activities competitively, because of its structural similarity With D-glucose. Trehalase from Nocardia sp., one of the most 1,5-AG-sensitive enzymes, was used in the determination of 1,5-AG concentration, which is a useful marker for the diagnosis of diabetes. A good linear relationship was observed between 1,5-AG concentration in the range of 0.02 to 1.0 mM and the extent of trehalase inhibition by 1,5-AG. The 1,5-AG concentration range could be determined by estimating enzymatically the amount of the reaction product, D-glucose, produced by the trehalase.