Binding of 4-methylumbelliferyl chitotetraoside ((GlcNAc)4-MeU) to hen lysozyme [EC 3.2.1.17] was studied by measuring changes in fluorescence at 375 nm. Hydrolysis of (GlcNAc)4-MeU catalyzed by lysozyme was studied by measuring the release of 4-methylumbelliferone from (GlcNAc)4-MeU fluorimetrically, and the kinetic constants were determined in the pH range of 2 to 8 at 0.1 ionic strength and 42 degrees C. The binding and kinetic data showed that (GlcNAc)4-MeU binds to subsites A to E (productive binding) and subsites A to D with the nonreducing sugar residue extending beyond subsite A (nonproductive binding). The fraction of the productive complex was 0.77 at pH 8.5. The pH dependence of the kinetic constants was analyzed assuming that the molecular species with ionized Asp 52 and protonated Glu 35 is active and Asp 101 participates in the binding (Phillips (1966) Sci. Am. 215, 78-90; Blake et al. (1967) Proc. Roy. Soc. B167, 378-388), and the pK values of these groups were determined. The pK values of Asp 52, Glu 35, and Asp 101 were 3.60, 6.20, and 4.20, respectively, for free lysozyme, 3.40, 6.55, and 3.40, respectively, for the productive complex, and 3.95, 6.55, and 3.30, respectively, for the nonproductive complex. The pK values for free lysozyme were in excellent agreement with those obtained by analysis of the kinetic constants for (GlcNAc)3-MeU (Yang & Hamaguchi (1980) J. Biochem. 87, 1003-1014). The free energy of activation was 24 kcal mol-1 at pH 5.2. Comparison with the corresponding value obtained for hydrolysis of (GlcNAc)6 (Banerjee et all (1975) J. Biol. Chem. 250, 4355-4367) suggests that the interactions of GlcNAc residues with subsites E and F in the transition state are important in lysozyme catalysis. Hydrolysis of (GlcNAc)2-MeU catalyzed by lysozyme was also studied, and the Kcat/Km values for (GlcNAc)2-MeU, (GlcNAc)3-MeU, and (GlcNAc)4-MeU were compared.