An intracellular hydrolase able to cleave l-lysine-p-nitroanilide was purified from Lactobacillus plantarum strain ESB5004 via two steps of precipitation with ammonium sulfate (at 30 and 50% (w/v)), followed by hydrophobic interaction and ion-exchange chromatographies. The aminopeptidase was purified up to 11-fold, with a final yield of ca. 1%. Its native molecular weight is ca. 70kDa, and it is apparently composed of two subunits, the molecular weight of which is 34kDa. The enzyme was assayed using a wide variety of p-nitroanilide (pNA) derivatives as substrates: it hydrolyzed preferentially pNA adducts of hydrophobic and basic amino acid residues; no hydrolysis was in particular observed of Glu-pNA, Gly-pNA or Pro-pNA. The enzyme activity was removed by the metal-chelating agent EDTA, thus suggesting that it is a metallo-enzyme; however, the EDTA-inhibited enzyme was reactivated in the presence of Co 2 + . Optimal aminopeptidase activity was obtained at 28 o C (pH 7.0) and pH 6.5 (37 o C). The enzyme was inhibited by 10mM CaCl 2 or MgCl 2 .