Two mutant strains of Escherichia coli BL21(DE3), called C41(DE3) and C43(DE3) and originally described by Miroux and Walker, are frequently used to overcome the toxicity associated with overexpressing recombinant proteins using the bacteriophage T7 RNA polymerase expression system. Even when the toxicity of the plasmids is so high that it prevents transformation in the strain BL21 (DE3), the toxic proteins can often be expressed successfully in C41 (DE3) and/or C43(DE3). In this work, using a range of plasmids coding for several types of proteins, we investigated in BL21(DE3), C41(DE3), and C43(DE3) their ability to undergo transformation and to express. While transformation was always possible in C41(DE3) and C43(DE3), we could not obtain transformants in BL21(DE3) for 62% of the expression vectors tested. Moreover, after induction, the expression of heterologous proteins in both mutant strains is generally better than in BL21(DE3). In this study, we also enhanced the stability of plasmids in culture during the expression of proteins by adding the par locus from the plasmid pSC101 to the vector backbone. The stability of a subset of the plasmids (measured 3 h after induction) was determined in C41 (DE3) and C43(DE3) and varies from 62 to 92% for C43(DE3) and from 10 to 90% for C41 (DE3). This study demonstrates the usefulness of these strains C41 (DE3) and C43(DE3) in solving the problem of plasmid instability during the expression of toxic recombinant proteins. (C) 2004 Elsevier Inc. All rights reserved.