The modes of action of two endo-1,4-beta-D-xylanases (X-I and X-II-B) from Aspergillus sojae were investigated using xylooligosaccharides and their corresponding alditols. Due to the dependence of the molecular activity (k(0)) on the degree of polymerization of substrates, it was suggested that each xylanase had five subsites. With regard to the bond-cleavage frequencies of various xylooligosaccharide-alditols, the catalytic site of each xylanase was located between the third and fourth subsites, counting from the terminal site, which was attached to the non-reducing end of the substrates. The intrinsic rate constants (k(int)) for the hydrolysis of glycosidic linkages of X-I and X-II-B were calculated to be 56 and 43 s(-1), respectively. Evaluation of subsite affinities of each xylanase : the total subsite affinity of (A(3) +A(4)), adjacent to the catalytic site of X-I, had a positive value (0.10 kcal/mol), whereas that of X-II-B had a negative value (-0.39 kcal/mol). The other subsite affinities of X-I exhibited higher values than that of X-II-B, except for the subsite affinity of A(1).