Solvent isotope effects have been examined for the action of the zinc-containing metalloenzyme carboxypeptidase A on ester and peptide substrates. The kinetic parameters for the carboxypeptidase-catalyzed hydrolysis of an ester, O-(trans-cinnamoyl)-L-β-phenyllactate, in 0.05 M Tris-DCl buffer containing 0.5 M NaCl at pD 8.07 and 25° were compared with those obtained from measurements done in 0.05 M Tris-HCl buffer containing 0.5 M NaCl at pH 7.52 and 25°. A (kcat)H2O/(kcat)D2O ratio of approximately 2 was obtained. The value of the Michaelis constant Km was unaffected by the change in solvent as was the inhibition constant, Ki, found for the product, L-β-phenyllactate, which is a competitive inhibitor. These results indicate that a catalytic step involving general base catalysis is probably important in the carboxypeptidase-catalyzed hydrolysis of an ester. A similar set of experiments carried out on the peptide substrate, N-(N-benzoylglycyl)-L-phenylalanine gave ambiguous results. The role of the zinc ion in the catalytic action of carboxypeptidase A can be considered in the light of these findings.