Fed-batch techniques were employed to obtain high cell density cultures (92–100 g DCW/L) of Escherichia coli strain X90 producing a recombinant serine protease, rat anionic trypsin, secreted to the periplasm. The specific growth rate was controlled to minimize growth-inhibiting acetate formation by utilizing an exponential feeding profile determined from mass balance equation. The volumetric yield of recombinant rat anionic trypsin was 56 mg/L, and the final cell density was 92 g DCW/L when the culture was induced in the late logarithmic phase. However, when the culture was induced in the early logarithmic phase, the volumetric yield was 13 mg/L and the final cell density was 14 g DCW/L. Thus, the induction timing is shown to have a significant effect on the final cell density as well as the overall volumetric yield of the recombinant protease. © 1993 Wiley & Sons, Inc.