Abstract The rate of lysozyme-catalyzed reaction of the β(1 → 4)-linked hexasaccharide of N-acetylglucosamine was determined at 40°, 0.1 ionic strength, and pH 2 to 10.5. Reaction rate was determined by measuring increase in reducing sugar for the hydrolysis reaction or by charcoal column analysis for the transglycosylation reaction. At all pH values, only one bond in the hexasaccharide is cleaved. Apparent first order kinetics is followed at substrate concentrations above Km, in accord with equally strong binding of hexasaccharide substrate and tetrasaccharide product. Measurements of the hydrolysis reaction above 10-4 m substrate were complicated by transglycosylation to the substrate as acceptor. The pH profile gave for κcat the kinetic apparent pK values 3.8 and 6.7 and a maximum value of κcat 0.15 s-1, and for κcat/Km the pK values 4.2 and 6.1. Equilibrium measurements of hexasaccharide binding at 40° and 0.1 ionic strength showed that Km equals Ks, with maximum value 9 x 10-6 m at pH 5. Detailed interpretation of the pH profiles was carried out under the assumptions that non-productive complexes are of kinetic importance and the structure of the productive complex and the catalytic mechanism are those suggested by the crystallography. This analysis gave for the free enzyme and nonproductive enzyme-hexamer complex, respectively, the values pK 6.1 and pK 6.7 for Glu-35 and pK 3.4 to 3.7 and pK 3.8 for Asp-52.