Exponentially fed-batch cultures (EFBCs), fed with medium containing a highly concentrated carbon source, are commonly employed for attainment of high cell densities. However; large variations in environmental conditions occur, and quasi-steady-state is usually achieved only for the limiting substrate concentration restricting the use of such cultures in kinetic characterization studies. In this work we report the production of recombinant penicillin acylase (PA) in EFBC of an E. coli JM101 transformed with the pPA102 plasmid, which includes the PA gene under regulation of the lacZ gene promoter and using isopropyl-beta-thio-galactopyranoside (IPTG) as inducer. The culture was fed with nonconcentrated complete medium, resulting in the attainment of quasi-steady-state conditions not only in substrate concentration, but also in cell concentration and in the specific rates of growth, product production, and substrate consumption. Similar transient behavior was observed between EFBC and chemostat results. At quasisteady-state, the dilution rate in the EFBC equaled the growth rate. Specific PA production rate during the fed-batch phase remained relatively constant at each dilution rate and followed typical Luedeking-Piret kinetics, with growth-associated and non-growth-associated constants of 142 U g(DCW)(-1) and 7.2 U g(DCW)(-1) h(-1), respectively. Specific glucose consumption rate linearly increased from 0.025 to 0.6 g g(DCW)(-1) h(-1) as the dilution rate increased from 0.01 to 0.35 h(-1). The maximum specific PA activity increased with decreasing dilution rate, reaching ifs highest value of 2.0 U mg(-1) at a dilution rate of 0.01 h(-1), the lowest dilution tested. Such an activity was 7-fold higher than in batch culture and, to our knowledge, represents the highest value reported in the literature.