Trehalase, an anomer-inverting glycosidase, hydrolyzes only alpha,alpha-trehalose in natural substrates to release equimolecular beta-glucose and alpha-glucose. Since the hydrolytic reaction is reversible, alpha,alpha-[1,1'-(2)H]trehalose is capable of synthesis from [1-(2)H]glucose through the reverse reaction of trehalase. alpha-Secondary deuterium kinetic isotope effects (alpha-SDKIEs) for the hydrolysis of synthesized alpha,alpha-[1,1'-(2)H]trehalose by honeybee trehalase were measured to examine the catalytic reaction mechanism. Relatively high k(H)/k(D) value of 1.53 for alpha-SDKIEs was observed. The data imply that the catalytic reaction of the trehalase occurs by the oxocarbenium ion intermediate mechanism. In addition, the hydrolytic reaction of glycosidase is discussed from the viewpoint of chemical reactivity for the hydrolysis of acetal in organic chemistry. As to the hydrolytic reaction mechanism of glycosidases, oxocarbenium ion intermediate and nucleophilic displacement mechanisms have been widely recognized, but it is pointed out for the first time that the former mechanism is rational and valid and generally the latter mechanism is unlikely to occur in the hydrolytic reaction of glycosidases.