Several beta-lactam acylases produced by different microorganisms (Escherichia coli, Kluyvera citrophila, Acetobacter turbidans, and Bacillus megaterium) have been evaluated as catalysts for the syntheses of relevant beta-lactam antibiotics (ampicillin, cephalexin, and cefamandole). These enzymes displayed very different synthetic properties showing large differences in synthetic yields (by a 4- to 5-fold factor) depending on the antibiotic and the enzyme. The enzyme from A. turbidans presented the best properties for the synthesis of ampicillin, which is a low activity in the hydrolysis of the antibiotic and a high specificity for the transformation of the ester into antibiotic. Although this enzyme was able to transform approximately 80% of phenylglycine methyl ester into ampicillin, it was unsuitable for the synthesis of cephalexin and cefamandole. In fact, all of the enzymes showed significant hydrolysis rates of the antibiotics compared to the synthetic activity, although the enzyme from E. coli exhibited the highest specificity for the transformation of eaters into these antibiotics. To prevent the hydrolysis of the antibiotic, a two-phase aqueous system was used to extract the antibiotic from the enzyme environment. In this way, high synthetic yields could be obtained, e.g. 80% of phenylglycine methyl ester was transformed into cephalexin using the enzyme from E. coli.