We studied high cell density continuous culture (HDCC) of a recombinant (E. coliHB101 (pPAKS2)) and a mutant (E. coli ATCC 11105) strains ofE. coli producing penicillin acylase(PA). Using pure oxygen, high cell density up to 95 g/l was obtained without significant inhibition by a main byproduct, acetic acid. The operation was simple and productivity was several times higher than those of conventional batch and continuous culture. Dissolved oxygen level and CO2 concentration were important variables, and glucose concentration was naturally regulated in HDCC.