The nature of chloride dependency on the activity of angiotensin I-converting enzyme which catalyses the release of C-terminal dipetide from angiotensin I decapeptide was studied by kinetic analysis using Hippuryl-Gly-Gly and N-Cbz-Gly-Gly-Pro as the substrate analogous. The angiotensin I-converting enzyme acts as a general dipeptidyl carboxypeptidase, but unlike other peptidase it shows a strong dependency on the presence of chloride ion for the full activity. When the activity of the enzyme was measured by a modified ninhydrin method employing an ion-exchange column of Aminex A-4, a sigmoidal curve of the enzyme activity profile was obtained as a function of chloride concentration. No change in the enzyme activity was found with other halogen ions such as fluoride, bromide, or iodide. The enzyme seems to be very specific toward chloride ion as a cofactor for the activity. When the enzyme activity was demonstrated by varying the concentration of the peptide substrate at different fixed concentrations of chloride ion, the resulting reciprocal plots showed a typical converging pattern indicating a possible involvement of a ternary complex between the enzyme, the peptide substrate and chloride ion. From the results it is suggested that chloride may increase the rate of enzyme reaction by decreasing the apparent $K_m$ value. The calculated values for HippurylGly-Gly and N-Cbz-Gly-Gly-Pro were 0.07 and 0.46, respectively. Based on the kinetic data obtained, it is suggested that chloride may have a role as an allosteric effector in the binding of the enzyme and substrate.