The stabilizing effect of calcium ions on thermolysin and Bacillus subtilis var. amylosacchariticus neutral protease has been investigated. Calcium and zinc ions were removed from the proteases by gel filtration over Sephadex G-25 equilibrated with metal chelating agents. Using these enzymes with different metal content, heat inactivation kinetics were studied at various temperatures. Removal of calcium ions caused a sharp decrease in thermostability and diminished the values of the activation enthalpy (deltaH*) and entropy (deltaS*) for heat inactivation. There was little difference in stability between thermolysin containing 0.3 g-atom/mol and B. subtilis neutral protease containing 1.4 g-atoms/mol. Calcium binding isotherms of the proteases were obtained by equilibrium gel chromatography with various concentrations of free calcium ions. Thermolysin had four independent calcium binding sites with an identical intrinsic binding constant (K) of 2.0 X 10(4) M-1. B. subtilis neutral protease had four independent sites. The K value for three sites was 1.1 X 10(5) M-1 and the binding constant for the other site was 1.5 X 10(3) M-1. There was little difference in total free energy change for calcium binding between these proteases. From these results it is concluded that the stabilizing effect of calcium on these enzymes is almost equal, and the extra thermal stability of thermolysin is likely to come from its polypeptide chain structure.