A medium for culturing Escherichia coli JM109 pcDNA3S as a DNA Vaccine was developed using a stoichiometric model. In batch fermentation, using the designed medium MWI and MW2, the plasmid DNA yield was 60 +/- 5.0 and 30 +/- 4.0 mg/l, respectively. However, plasmid the DNA yield was only 4 +/- 0.4 mg/l in LB medium and 20 +/- 3.5 mg/l in LEG medium. The plasmid stability characteristics of the JM109 pcDNA3S grown in LB, LEG, MWI and MW2 were determined in continuous cultures. After 96 h cultivation, the plasmid stability was 11.3% higher when bacteria were cultivated in MW1 medium relative to cell growth in MW2 medium, 30.6% higher relative to cell growth in LB medium, 63.3% higher relative to cell growth in LEG medium. These results show that amino acids and nucleosides affect plasmid DNA yield significantly. It seems that the defined medium can support much higher plasmid DNA yield than a complex medium. Amino acids and nucleosides are important factors to be considered during production of plasmid DNA.