The kinetics of the asymmetric hydrolysis of N-acetyl-DL-butyrine catalyzed by L-aminoacylase to obtain optically pure L-butyrine is described. Some of the constants are determined from the initial reaction rates and others from long-term experiments in batch reactors by the numerical integration of the reactor design equation and minimization of the kinetic parameters. The methodology described can be applied to the kinetic study of other complex biocatalytic systems. Studies on enzyme activation by adding different divalent metal ions and enzymatic deactivation are also included.