Recombinant Escherichia coli cells with high penicillin acylase (PA) activity were immobilized by gel entrapment with agar. This biocatalyst was used to study the effect of pH on the synthesis of ampicillin from phenylglycine methylester (PGME) and 6-aminopenicillanic acid (6-APA). The parallel hydrolysis reactions of PGME and ampicillin were also studied. A selective inhibition of the hydrolysis of the ester was possible by controlling the pH at 6.0. At such conditions, and using 6-APA solutions ranging from 50-200 mM, a 75% conversion to ampicillin was obtained. This yield was higher than obtained with other strategies. The reaction kinetics was described by a second-order model for ampicillin synthesis with experimentally determined Michaelis-Menten constants of 27 and 25 mM for 6-APA nd PGME, respectively. In addition, ampicillin and PGME were hydrolyzed by the enzyme following Michaelis-Menten kinetics with K-m values of 40.5 and 30 mM, respectively. A good correlation was found between experimental results of synthesis reactions and the kinetic model derived from initial rate experiments with only slight deviations at high substrate concentrations. This is the first report where the specific effect of pH on the synthesis of ampicillin was studied in detail. It is shown that, by controlling the pH, it is possible to inhibit the lateral undesirable reactions increasing the yield of the main reaction.