The penicillin acylase-catalyzed synthesis of ampicillin by acyl transfer from D-H-phenylglycine amide (D-PGA) to 6-aminopenicillanic acid (6-APA) becomes more effective when a judiciously chosen pH gradient is applied in the course of the process. This reaction concept is based on two experimental observations: 1) The ratio of the initial synthesis and hydrolysis rates (VsNH) is pH-dependent and exhibits a maximum at pH 6.5-7.0 for a saturated solution of 6APA; 2) at a fixed 6-APA concentration below saturation, VsNH increases with decreasing pH. Optimum synthetic efficiency could, therefore, be achieved by starting with a concentrated 6-APA solution at pH 7 and gradually decreasing the pH to 6.3 in the course of 6-APA consumption. A conversion of 96% of 6-APA and 71% of DPGA into ampicillin was accomplished in an opitimized procedure, which significantly exceeds the efficiency of enzymatic synthesis performed at a constant pH of either 7.0 or 6.3.