High-density culture was achieved through controlling specific growth rate by limiting glucose concentration to < 0.2gL(-1). Carboxylesterase B I capable of hydrolyzing organophosphate esters was purified from Escherichia coli strain BL21 carrying a cloned esterase B I gene from mosquito. The recombinant strain BL21 carrying pET-ESTB1 was used for the fermentation. Product formation was induced by either a temperature shift from 30 to 42 degrees C or by feeding a mixture of glucose and lactose. Cell growth and production of detoxifying enzyme were affected by oxygen availability. The maximum biomass of E. coli BL21 (pET-ESTB1) increased from 14.9 to 31.5 g dry cell weight l(-1). Using the host strain E. coli BL21 (DE3), detoxifying enzyme was over-expressed at a biomass level of up to 31.5 g dry weight l(-1). The enzyme had a molecular mass of 64 kDa, its optimum temperature was approx. 37 degrees C; at pH 7 the relative activity after 3 h was 85.9% at 28 degrees C, 64.9% at 34 degrees C, and 4.5% at 40 degrees C. The enzyme activity of cells grown at lower temperatures was much higher; at 18 degrees C it almost twice than at 20 or 22 degrees C. degrees 2006 Elsevier Ltd. All rights reserved.