Penicillin G (Pen-G) was hydrolyzed to 6-aminopenicillanic acid (6-APA) and phenylacetic acid (PAA) in a chromatographic reactor-separator using the mixture of immobilized Escherichia coli cells and a macroporous adsorbent as stationary phase and a phosphate buffer of pH 7.8 as eluant. Pen-G conversion of 98% was observed without adjustment of the eluant pH due to the effective separation of 6-APA from Pen-G and PAA. At a sample load of 600 mg Pen-G, the volume overload gave higher Pen-G conversion (86%) than the mass overload (68%), while their difference in product resolution (0.9 and 1.0, respectively) was insignificant.