The kinetics of the N-acetyl-L-norvaline methyl ester (non-specific substrate) hydrolysis by trypsin was studied within a wide substrate concentration range (0.02 mM--25.5 mM). The dependence of the initial reaction rate on the substrate concentration obeyed the Michaelis--Menten equation only at low (less than 0.3 mM) concentrations of the substrate. At higher substrate concentrations the reaction rate considerably exceeded the maximal rate determined from the Michaelis--Menten equation. The reaction kinetics at high substrate concentrations was in good agreement with the scheme for the substrate activation of the enzyme proposed by Trowbridge et al. The reaction product, N-acetyl-L-norvaline, did not activate the substrate hydrolysis by trypsin. The kinetic and activation parameters of the reaction (kcat = 0.0184 s-1, Km = 0.08 mM and kcat = 2.97 s-1 and Km = 56 mM, respectively, pH 8.0, 25 degrees) were determined.