Calcium is required for the stabilization of a-amylase because of primary binding (essential binding), but has been shown to inhibit hydrolytic catalysis due to secondary binding at the catalytic site in the enzyme. The role of calcium in the hydrolysis of soluble amylose by Bacillus amyloliquefaciens alpha-amylase was characterized using the equilibrium dissociation constant (K-m) and k(cat) for the hydrolytic catalysis. The enzymatic hydrolysis was inhibited by a relatively high concentration of calcium ions (\Ca2+\ greater than or equal to 2.0 mM). The dissociation constant (K-m) was increased with increasing calcium ion concentration. Because k(cat) was practically constant at the high calcium concentration range, a competitive inhibition kinetic model was applied to calculate the inhibition parameters in terms of the secondary calcium binding to the a-amylase. The enthalpy and entropy changes for the secondary binding were 54.8 kJ/mol and 215 J/mol (.) K, respectively, and these values suggest a strong entropic affinity for the bivalent ion binding to the enzyme. The thermodynamical analysis clearly shows the conformational changes in this alpha-amylase during the primary and secondary calcium ion binding.