The equilibrium constant for penicillin amidase-catalyzed hydrolysis of benzylpenicillin (Keq = 3.00 .+-. 0.24 .times. 10-3 m at pH 5.0) and the ionization constants for phenylacetic acid (PAA) and the amino grops of 6-aminopenicillanic acid (6-APA) were determined (4.20 and 4.60 under conditions of the kinetic experiments, respectively). The experimental data at pH 6.0 satisfactorily correlated with the theoretical pH-dependence for Keq constructed according to the hypothesis that benzylpenicillin synthesis has a thermodynamic optimum at pH 4.4 equal to a half-sum of the pK values for the carboxylic and amino groups of the PAA and 6-APA, respectively.