A simple novel method was introduced for determination of an inhibitor binding constant (Ki) and enthalpy of binding by isothermal titration microcalorimetry technique. This method was applied to the binding of fluoride ion, as an inhibitor, with the active sites of jack bean urease at pH = 7.0 (Tris 30 mM) and T = 300 degrees K. The dissociation equilibrium constant measured by this method was markedly consistent with the inhibition constant obtained from assay of enzyme activity in the presence of fluoride ion.