A methotrexate-resistant strain of Escherichia coli Type I produced exceptionally high levels of the enzyme dihydrofolate reductase (EC 1.5.1.3) in 6 h of batch fermentation. The culture had markedly improved performance under chemostat culture conditions in terms of enzyme yield and output. Temperature, pH, dilution rates, and nutrient composition were optimized under chemostat culture conditions. A maximum enzyme yield of 10,360 U l-1 and a specific activity of 22.84 U mg-1 were obtained under chemostat conditions at pH 7.0, temperature 37 degrees C, and dilution rate 0.2 h-1, using media containing 1.0 and 0.6% (w/v) dextrose and yeast extract, respectively. The culture's performance and enzyme yields in chemostat and the feasibility of large-scale production are discussed.