Hydrolysis of probe substrates, eight possible monodeoxy and mono-O-methyl analogs of p-nitrophenyl alpha-D-glucopyranoside (pNP alpha-D-Glc), modified at the C-2, C-3, C-4, and C-6 positions, was studied as part of investigations into the glycon specificities of seven alpha-glucosidases (EC 3.2.1.20) isolated from Saccharomyces cerevisiae, Bacillus stearothermophilus, honeybee (two enzymes), sugar beet, flint corn, and Aspergillus niger. The glucosidases from sugar beet, flint corn, and A. niger were found to hydrolyze the 2-deoxy analogs with substantially higher activities than against pNP alpha-D-Glc. Moreover, the flint corn and A. niger enzymes showed hydrolyzing activities, although low, for the 3-deoxy analog. The other four alpha-glucosidases did not exhibit any activities for either the 2- or the 3-deoxy analogs. None of the seven enzymes exhibited any activities toward the 4-deoxy, 6-deoxy, or any of the methoxy analogs. The hydrolysis results, with the deoxy substrate analogs, demonstrated that alpha-glucosidases having remarkably different glycon specificities exist in nature. Further insight into the hydrolysis of deoxyglycosides was obtained by determining the kinetic parameters (k(cat) and K(m)) for the reactions of sugar beet, flint corn, and A. niger enzymes.