The action pattern and reaction mechanism of the exo-beta-(1,4)-galactanase from Aspergillus niger var. aculeatus on galactooligosaccharides of degree of polymerization (dp) from 2-6 were investigated. The reaction was assayed at the optimum pH of both hydrolysis and transglycosylation, and HPLC analysis of the reaction mixtures was used to obtain kinetic parameters. Hydrolysis of oligosaccharides to galactose followed a Michaelis-Menten behavior whatever the dp. Catalytic activity was always higher for hydrolysis than for transglycosylation. At the optimum pH, the affinity of the enzyme increased for hydrolysis and decreased for transglycosylation with increasing dp. Transglycosylation was explained as a dependent binding of two similar molecules of substrates. The dependence of the kinetic parameters on the degree of polymerization of substrates was used to evaluate the subsite affinities. The active site of exogalactanase was concluded to possess three subsites with the catalytic site being located between subsites 1 and 2.