To improve the plasmid stability during the production of antihuman ovarian carcinoma x antihuman CD3 single-chain bispecific antibody (AhOCxAhCD3), the Escherichia coli BL21(DE3) host cell was optimized serially. Firstly, an isogenic recombination-deficient (recA(-)) derivative of BL21(DE3), namely BLR(DE3), was used as host instead of BL21(DE3). Although the segregational plasmid stability was greatly improved, AhOCxAhCD3 yield was not improved due to the severe growth inhibition of plasmid-bearing BLR(DE3) cells and the competitive plasmid instability after induction. Secondly, a mutant BLR(DE3), namely BLRM(DE3), was screened by using LB agar plates plus ampicillin and isopropyl-beta-D-thiogalactopyranoside. Using this new host, growth inhibition of recombinant cells after induction was eliminated, and plasmids could be stably maintained even after long-time induction in a nonselective medium. At last, about 1.2 g/l AhOCxAhCD3, which was about thrice as much as those of recombinant BL21(DE3) and BLR(DE3) strains, was yielded.