Substrate binding subsites of barley chitinase and goose egg white lysozyme were comparatively investigated by kinetic analysis using N-acetylglucosamine oligosaccharide as the substrate. The enzymatic hydrolysis of hexasaccharide was monitored by HPLC, and the reaction time-course was analyzed by the mathematical model, in which six binding subsites (B, C, D, E, F, and G) and bond cleavage between sites D and E are postulated. In this model, all of the possible binding modes of substrate and products are taken into consideration assuming a rapid equilibrium in the oligosaccharide binding processes. To estimate the binding free energy changes of the subsites, time-course calculation was repeated with changing the free energy values of individual subsites, until the calculated time-course was sufficiently fitted to the experimental one. The binding free energy changes of the six binding subsites, B, C, D, E, F and G, which could give a calculated time-course best fitted to the experimental, were 0.0, -5.0, +4.1, -0.5, -3.8, and -2.0 kcal/mol for barley chitinase, and -0.5, -2.2, +4.2, -1.5, -2.6, and -2.8 kcal/mol for goose egg white lysozyme. The binding mode predicted from the p-nitrophenyl-penta-N-acetylchitopentaoside splitting pattern for each enzyme was also analyzed by the identical subsite model. Using the free energy values listed above, the binding mode distribution calculated was fitted to the experimental with a slight modification of free energy value at site G. We concluded that the binding subsite model described above reflects the substantial mechanism of substrate binding for both enzymes. The relatively large disparity in free energy value at site C between these enzymes may be due to the different secondary structures of polypeptide segments interacting with the sugar residue at site C.