α-Amylase from Sorghum bicolor, is reversibly unfolded by chemical denaturants at pH 7.0 in 50mM Hepes containing 13.6mM calcium and 15mM DTT. The isothermal equilibrium unfolding at 27°C is characterized by two state transition with ΔG (H 2 O) of 16.5kJ mol −1 and 22kJ mol −1 , respectively, at pH 4.8 and pH 7.0 for GuHCl and ΔG (H 2 O) of 25.2kJ mol −1 at pH 4.8 for urea. The conformational stability indicators such as the change in excess heat capacity (ΔC p ), the unfolding enthalpy (H g ) and the temperature at ΔG=0 (T g ) are 17.9±0.7kJ mol −1 K −1 , 501.2±18.2kJ mol −1 and 337.3±6.9K at pH 4.8 and 14.3±0.5kJ mol −1 K −1 , 509.3±21.7kJ mol −1 and 345.4±4.8K at pH 7.0, respectively. The reactivity of the conserved cysteine residues, during unfolding, indicates that unfolding starts from the ‘B’ domain of the enzyme. The oxidation of cysteine residues, during unfolding, can be prevented by the addition of DTT. The conserved cysteine residues are essential for enzyme activity but not for the secondary and tertiary fold acquired during refolding of the denatured enzyme. The pH dependent stability described by ΔG (H 2 O) and the effect of salt on urea induced unfolding confirm the role of electrostatic interactions in enzyme stability.