The Michaelis constant (K-m) and molecular activity (k(0)) of an exo-1,4-beta-glucosidase (EC 3.2.1.74) from Acetobacter xylinum subsp. sucrofermentans BPR2001 for hydrolysis of cello-oligosaccharides (G2-G6) were determined by steady-state kinetic analysis. The 1/K-m and k(0) values for G2 were much lower than those for G3-G6. The enzyme was competitively inhibited by glucono-delta-lactone and conduritol-beta epoxide. Based on the theory of Hiromi et al. (Biochim. Biophys. Acta, 302 : 362-375, 1973), the subsite affinities (A(i), i = 1-6) and the intrinsic hydrolysis rate constant for substrate linkage in a productive complex (ki,,) of the enzyme were kinetically estimated : A(1) = 2.46 kcal/mol, A(2) = - 0.44 kcal/mol, A(3) = 3.70 kcal/mol, A(4) = 0.33 kcal/mol, A(5) = 0.27 kcal/mol, A(6) = 0.06 kcal/mol, and k(int) = 33.4 s(-1). The subsite affinities were different from those for the beta-glucosidase from Aspergillus niger and for the exo-1,4-beta-glucosidase from Torulopsis wickerhamii, in that the Az value for the Acetobacter enzyme was negative. These results suggest that the enzyme possesses subsite affinities which have never previously been reported among exo-type glucosidases.