Nattokinase is a potent fibrinolytic enzyme. The composition of the medium for the production of nattokinase was optimized with a stepwise strategy. Effects of various nitrogen and carbon sources were investigated. Fractional factorial design (FFD) was applied to elucidate the key ingredients in the media and the results indicated that the soy peptone, yeast extract and calcium chloride have a significant effect on nattokinase production. Central composite design (CCD) was employed to search for the optimal concentration of the three key components and the experimental results were fitted with a second-order polynomial model at 98% level (P < 0.02). Canonical analysis results showed that the response surface had a maximal point. According to the model, the predicted maximal nattokinase volumetric concentration was about 1273.8 units/ml and the corresponding concentration of soy peptone, calcium chloride and yeast extract were around 8.28, 0.64 and 0.74 g/l, respectively. The trial checking the developed medium showed a high nattokinase activity of 1300 60 units/ml. (c) 2005 Elsevier Ltd. All rights reserved.