This study was carried out to determine the optimal folding condition of alpha-amylase from Bacillus megaterium WHO using response surface methodology (RSM). A first-order model showed that three factors namely glycerol, Ca2+ and protein concentration had the most significant effect on refolding. Analysis of the results showed that glycerol was better than the other polyols due to its effect on protein stability. Since a-amylases are known to contain calcium ions in their structure, the presence of calcium in the refolding buffer was compulsory. The concentration of protein had the most significant quadratic effect on the response studied. A second-order polynomial model was developed to quantify the relationships between variables. It was shown that the combination of 20% (v/v) glycerol, 25 mM Ca2+ and 0.3 (mg/ml) protein was the most efficient condition for in vitro refolding of a-amylase. Under the optimal condition the yield of refolding was enhanced up to 7-fold. In order to analysis the size distribution in optimized and basic medium, dynamic light scattering (DLS) was fulfilled. The information gathered in this study showed that the use of solvent engineering and optimization procedure can be a general method for protein refolding. (c) 2012 Elsevier Inc. All rights reserved.