An enzyme, carboxypeptidase CN, was separated from the exocarp of Citrus natsudaidai HAYATA. The enzyme was homogeneous in disc electrophoresis and ultra-centrifugal analysis. It liberated neutral, acidic, and basic amino acids including proline from the C-termini of N-substituted dipeptides at varying rates. Of the substrates tested, Z-Gly-Pro, Z-Pro-Pro, and Z-Glu-Pro were hydrolyzed very slowly. The enzyme hydrolyzed Z-Gly-Pro-Leu-Gly, the oxidized B chain of bovine insulin, and bradykinin potentiator C sequentially from their C-termini. The specific activity of the purified enzyme was 65.4 units per mg of protein for Z-Glu-Phe. It possessed weak esterase activity, but was free from endopeptidase and aminopeptidase activities. It had a pH optimum at pH 5.5 for Z-Glu-Phe. It was strongly inhibited by HgCl2 and DFP, but not by other metal ions, anions, EDTA, or o-phenanthroline. NEM, PCMB, DTT, and MIA showed no significant effect on the enzymatic activity at pH 5.5. Slow inactivation, however, occurred when the enzyme was preincubated with PCMB at pH 7.0. The values of s20, w and D20,w were 5.5 S and 5.5×10−7cm2 per sec, respectively, and the molecular weight was calculated to be 93,000 from these values.