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 (k(0)) 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; A(1)=2.93 kcal/mol, A(2)=3.67 kcal/mol, A(3)=0.64 kcal/mol, A(4)=0.12 kcal/mol, A(5)=-0.07 kcal/mol, A(6)=-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 Al were much larger than those for A(3).