BACKGROUND: The proteases are among the most important groups of enzymes. Therefore, it is important to produce inexpensive and optimized media for large-scale commercial production. In the present work, three different Shewanella species were screened on skim milk agar medium for their ability to produce alkaline protease. The effects of different culture conditions were optimized for alkaline protease production by S. oneidensis MR-1 using a Box-Behnken design combined with response surface methodology (RSM). RESULTS: Highest yield (112.90 U mL(-1)) of protease production was obtained at pH 9.0, a temperature of 30 degrees C, glucose (12.5 g L-1), tryptone (112.5 g L-1) and an incubation period of 36 h. A second-order polynomial regression model was used for analysis of the experiment. The experimental values were in good agreement with predicted values, with correlation coefficient 0.9996. CONCLUSION: Carbon and nitrogen, pH, temperature and incubation period were chosen as the main factors to be used in an experimental design for optimization to produce low-cost enzymes, potentially for use on an industrial scale. A 60% increase in enzyme activity was achieved in the optimized medium compared with the original medium. (C) 2008 Society of Chemical Industry