A high-expression system for penicillin G acylase (PGA) was developed using the high PGA-producing strain Escherichia coli RE3 as a host and a recombinant plasmid pKA18 which was constructed by cloning the chromosomal pga gene coding for PGA in the strain RE3 on multicopy vector pK19. One particular objective for the elaboration of this expression system was the selection of a convenient host strain and modification of the growth conditions. From a total of 15 hosts, the strain RE3 cultured in a mineral medium exhibited the highest expression of a pga-encoding gene from the recombinant plasmid and high segregational plasmid stability. The high-expression system led to an increase in the specific activity of PGA up to 1,000 U g(-1) cell dry weight and a total activity of about 4,500 U l(-1) of the culture in a laboratory fermentor.