The subsite affinities of beta-glucosidase (EC 3.2.1.21) with high beta-xylosidase activity from Aspergillus sojae on various xylooligosaccharides (degree of polymerization: n = 2-6) were investigated by steady-state kinetic analysis. The molecular activity (k0) value of the enzyme for xylobiose was not markedly different from those of other substrates (n = 3-6). The arrangement of the subsite affinities (A(i), i = 1-6) was evaluated; A1 = 2.93 kcal/mol, A2 = 3.67 kcal/mol, A3 = 0.64 kcal/mol, A4 = 0.12 kcal/mol, A5 = -0.07 kcal/mol, A6 = -0.05 kcal/mol, and the intrinsic rate constant (K(int)) was 7.6 s(-1). The subsite structure was similar to those of beta-glucosidase from A. niger and alpha-glucosidases from A. niger and Mucor javanicus, where the values for A1 were much larger than those for A3.