A hydrolase activity that cleaves lysyl-p-nitroanilide (Lys-pNA) has been purified from the cytoplasm of Lactococcus lactis subsp. cremoris AM2 by chromatography on DE52, DEAE Affi-Gel Blue Gel, Hydroxyapatite Bio-Gel HTP and Phenyl Sepharose. The purified aminopeptidase was found to have a native Mr of 50 000–55 000 by gel filtration chromatography and by FPLC gel filtration on Superose 12 and to be composed of a single polypeptide chain following SDS-PAGE. Enzyme activity was almost completely inhibited by EDTA, amastatin, puromycin and bestatin, while the sulphydryl-reactive agents p-chloromercuribenzoate and iodoacetamide were inhibitory. The enzyme was found to be very unstable during the purification procedures at 4°C and its stability was greatly improved when 10 ml glycerol/l and 2 mm-dithiothreitol were included in the purification buffers. The purified enzyme was found to hydrolyse a wide range of dipeptides, tripeptides and longer peptides provided that proline was not present in the penultimate position from the N-terminus or that a pyroglutamyl residue was not present at the N-terminus. While neither Asp-pNA nor Pro-pNA was hydrolysed by the purified enzyme, the release of N-terminal acidic residues from peptides was observed in addition to the release of N-terminal proline from Pro–Leu–Gly–NH2, Pro–Leu–Gly–Gly and Pro–His–Pro–Phe–His–Leu–Phe–Val–Tyr. This ability of Lys-pNA hydrolase to release N-terminal proline residues was employed in concert with a purified aminopeptidase P preparation to release alternate N-terminal amino acids from Tyr–Pro–Phe–Pro–Gly. The complementary action of these enzymes represents an alternative mechanism to that of post-proline dipeptidyl aminopeptidase for metabolism of proline-containing peptides.