BACKGROUND: The culture medium and fermentation conditions for the production of constitutive chitosanase from a newly isolated Bacillus sp. RKY3 were optimized statistically. RESULTS: The variables significantly influencing both chitosanase production and cell growth were screened through the Plackett-Burman design, by which maltose, beef extract, MgSO4, and incubation time were identified as the most significant variables. The optimum values of the selected variables and their mutual interactions were determined through the steepest ascent method and Box-Behnken experimental design. The results demonstrated that 62.30 U mL(-1) chitosanase activity was predicted with optimum conditions of maltose (30.18 g L-1), beef extract (15.25 g L-1), MgSO4 (0.26 g L-1), and incubation time (50.02 h). The predicted response was verified by the validation experiments, and the optimum conditions resulted in a maximum chitosanase activity of 63.53 +/- 1.22 U mL(-1). CONCLUSION:The optimization of fermentation variables resulted in an approximately 11.3-fold increase in chitosanase activity compared with that observed under unoptimized conditions (from 5.63 U mL(-1) to 63.53 U mL(-1)). (c) 2009 Society of Chemical Industry