Abstract Aqualysin I is a heat-stable protease; in the presence of 1 mM Ca 2+, the enzyme is stable at 80°C and shows the highest activity at the same temperature. After gel filtration to remove free Ca 2+ from the purified enzyme sample, the enzyme (holo-aqualysin I) still bound Ca 2+ (1 mol/mol of the enzyme), but was no longer stable at 80°C. On treatment of the holo-enzyme with EDTA, bound Ca 2+ decreased to about 0.3 mol/mol of the enzyme. The thermostability of holo-aqualysin I was dependent on the concentration of added Ca 2+, and 1 mM added Ca 2+ stabilized the enzyme completely, suggesting that aqualysin I has at least two Ca 2+ binding sites, i.e. stronger and weaker binding ones. Titration calorimetry showed single binding of Ca 2+ to the holo-enzyme with an association constant of 3.1×10 3 M −1, and Δ H and TΔ S were calculated to be 2.3 and 6.9 kcal/mol, respectively, at 13°C. La 3+, Sr 2+, Nd 3+, and Tb 3+ stabilized the holo-enzyme at 80°C, as Ca 2+ did. These results suggest that the weaker binding site exhibits structural flexibility to bind several metal cations different in size and valency, and that the metal binding to the weaker binding site is essential for the thermostability of aqualysin I.