The high activity of T7 RNA polymerase has made the T7 RNA polymerase-based expression system very powerful for highlevel expression of recombinant protein. However, the overactivity of T7 RNA polymerase would also bring about negative effects on plasmid stability and protein production, especially when expressing a toxic protein. If the latter role is dominant, it is necessary to adopt some measures to attenuate the activity or the amount of T7 RNA polymerase in the cells. Apart from the stringent regulation by inserting some genes reducing the amount or the activity of T7 RNA polymerase into plasmids, optimizing the culture conditions would be another way. In this work, we have studied the effects of various culture conditions on the plasmid stability and the target protein yield including selective pressure, culture temperature, toxicity of the target protein and the catabolite repression caused by glucose. The results have indicated that adding antibiotic after induction has little effect in increasing plasmid stability, but inducing expression at low temperature and adding glucose to the medium improved the plasmid stability and the protein yield to a large extent. (C) 2003 Elsevier Science (USA). All rights reserved.