Optimal concentrations of glucose, yeast extract and nutrient broth for the production of beta-lactamase by B. subtilis were predicted using a statistical experimental design and then tested. More yeast extract (13 vs. 1 g/L), less glucose (7.4 vs. 10 g/L), and less nutrient broth (12.6 vs. 15 g/L) were required to achieve high beta-lactamase activities (5700 U/L) instead of high cell growth rates (1.2 h(-1)).