The cultivation conditions for N176 cephalosporin acylase production were optimized using Escherichia coli JM109/pCK305S, which encodes a highly expressed mutant, C305S, in which Cys(305) is altered to Ser, under the control of the trp promoter. Processing of the precursor protein to a two-chain active form in vivo was a key factor for high-level production of the enzyme. The active acylase was produced in a high amount under the following conditions : (i) addition of Trp in the second seed medium, (ii) the use of glycerol as a carbon source, (iii) induction with IAA at 9 h, and (iv) a shift down in the cultivation temperature from 25 to 22.5 degrees C after induction. Under these high-level production conditions, the OD at 600 nm was found to increase in proportion to the number of colony forming units (CFUs), indicating that the CFU is an important index for the expression of the active enzyme.