An extracellular carboxypeptidase with a pH optimum of between 4 and 5 has been isolated from the culture medium of Penicillium janthinellum. Like penicillopepsin, this enzyme is only released when the stationary phase of growth is reached. The enzyme has been purified by affinity chromatography on phenylbutylamine-Sepharose followed by isoelectric focusing. The enzyme was found to be present in two forms with isoelectric points of 3.70 and 3.77. It has a molecular weight of about 48 000 and is inhibited by 10−7 M p-hydroxymercury benzoic acid. It is not inhibited by the metal chelators EDTA, o-phenanthroline, and 8-hydroxyquinoline, or by diisopropyl phosphorofluoridate. The presence of a single cysteine residue has been demonstrated by labelling the enzyme with 14C-iodoacetic acid. The action of the enzyme on glucagon, the S-sulfo-B-chain of insulin, and on penicillopepsin at pH 4.7 shows that it is a nonspecific carboxypeptidase and releases C-terminal proline, lysine, and arginine, as well as the other amino acids. Glycine appears to be the slowest residue to be released. Carbobenzoxy-L-glutamyl-L-tyrosine, the substrate used for routine assays, is hydrolyzed very rapidly. The enzyme also slowly hydrolyzed Leu–Phe, and splits glycine from Leu–Gly–Gly.A second enzyme with carbobenzoxy-L-glutamyl-L-tyrosine activity is present in the growth medium. It has an isoelectric point of 4.72 on an isoelectric focusing column. Preliminary inhibition studies of a partially purified preparation suggest that it differs from the other enzyme.