Media composition and culture conditions for surfactant stable alkaline protease production by Bacillus mojavensis A21 were optimized using two statistical methods. Plackett-Burman design was applied to find the optimal ingredients and conditions to improve yields. Response surface methodology (RSM), including central composite design, was used to determine the optimal concentrations and conditions. The results indicated that several components, including hulled grain of wheat (HGW), sardinella peptone (SP), NaCI, CaCl2, MgSO4, K2HPO4, KH2PO4, agitation, culture temperature and initial medium pH, had significant effects on production. The statistical model was constructed via central composite design (CCD) using four selected variables (HGW, NaCl, KH2PO4 and K2HPO4). Under the proposed optimized conditions, the protease experimental yield (1860.63 U/mL) closely matched the yield predicted by the statistical model (1838.60 U/mL) with R-2 = 0.98. An overall 14.0-fold increase in protease production was achieved using the optimized medium (HGW 30.0 g/L, SP 1.0 g/L, NaCI 2.0 g/L, KH2PO4 1.0 g/L, K2HPO4 0.3 g/L, CaCl2 2.0 g/L, MgSO4 1.0 g/L and pH 9.0, compared with the unoptimized basal medium (starch 10.0 g/L, yeast extract 2.0 g/L, KH2PO4 0.1 g/L, K2HPO4 0.1 g/L, CaCl2 0.5 g/L and pH 8.0; 137 U/mL). A successful and significant improvement (14-fold) in the production of protease by the A21 strain was accomplished using cheap carbon and nitrogen substrates (HGW and SP), which may result in a significant reduction in the cost of medium constituents. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.