Penicillin amidases (PAs) from E. coli and A. faecalis are periplasmic enzymes that contain one tightly bound Ca2+ per molecule that does not directly participate in the enzymatic function. This ion may, however, be required for the maturation of the prepro-enzyme. The pro-enzyme of homologous PAs are translocated through the Tat(E. coli PA(EC)) and Sec- (A. faecalis PA(AF)) transport systems, respectively. Cell fractionation, electrophoresis, immunoblotting, and activity staining demonstrated that Ca2+ binding is required for the membrane transport and maturation of the pro-enzyme to active enzyme. Pro-enzyme without Ca2+ was targeted to the membrane but not translocated. Influence of Ca2+ in medium and feed was studied for high cell density cultivations of E. coli expressing these enzymes. Without Ca2+ in the feed the synthesis of the pre-pro-enzyme was hardly influenced. At optimal Ca2+ content in the feed the active enzyme amount could be increased by 2 orders of magnitude up to 0.9 g/L (PAEc) and 2.3 g/L (PA(AF)) or 4% (PA(EC)) and 8% (PA(AF)) of the cell dry weight. The corresponding specific activities are 1700 U (PA(EC)) and 14000 U (PA(AF)) per gram cell dry weight, respectively. These values are higher than those published previously. Thus, for optimal yields of the studied and other extra- and periplasmic enzymes that require Ca2+ or other ions as cofactors for membrane transport and maturation, sufficient cofactor must be added in the feed.