Abstract The four possible monodeoxy derivatives of p-nitrophenyl (PNP) α- d-galactopyranoside were synthesized, and hydrolytic activities of the α-galactosidase of green coffee bean, Mortierella vinacea and Aspergillus niger against them were elucidated. The 2- and 6-deoxy substrates were hydrolyzed by the enzymes from green coffee bean and M. vinacea, while they scarcely acted on the 3- and 4-deoxy compounds. On the other hand, A. niger α-galactosidase hydrolyzed only the 2-deoxy compound in these deoxy substrates, and the activity was very high. These results indicate that the presence of two hydroxyl groups (OH-3 and -4) is essential for the compounds to act as substrates for the enzymes of green coffee bean and M. vinacea, while the three hydroxyl groups (OH-3, -4, and -6) are necessary for the activity of the A. niger enzyme. The kinetic parameters ( K m and V max) of the enzymes for the hydrolysis of PNP α- d-galactopyranoside and its deoxy derivatives were obtained from kinetic studies.