The proteolytic system of lactic acid bacteria is essential for bacterial growth in milk but also for the development of the organoleptic properties of dairy products. Streptococcus thermophilus is widely used in the dairy industry.In comparison with the model lactic acid bacteria Lactococcus lactis, S. thermophilus possesses two additional peptidases (an oligopeptidase and the aminopeptidase PepS). To understand how S. thermophilus grows in milk, we purified and characterized this aminopeptidase. PepS is a monomeric metallopeptidase of <45 kDa with optimal activity in the range pH 7.5±8.5 and at 55 8C on Arg-paranitroanilide as substrate. PepS exhibits a high specificity towards peptides possessing arginine or aromatic amino acids at the N-terminus. From the N-terminal protein sequence of PepS, we deduced degenerate oligonucleotides and amplified the corresponding gene by successive PCR reactions. The deduced amino-acid sequence of the PepS gene has high identity (40±50%) with the aminopeptidase T family from thermophilic and extremophilic bacteria; we thus propose the classification of PepS from S. thermophilus as a new member of this family. In view of its substrate specificity, PepS could be involved both in bacterial growth by supplying amino acids, and in the development of dairy products' flavour, by hydrolysing bitter peptides and liberating aromatic amino acids which are important precursors of aroma compounds.Keywords: aminopeptidase T family; lactic acid bacteria; peptidase; Streptococcus thermophilus. Aminopeptidases (EC 3.4.11) are ubiquitous enzymes, frequently observed in animals, plants and microorganisms. As exopeptidases, they catalyse the release of free amino acids from peptides. They are involved in many different functions in the cell, such as protein maturation, protein turnover, hydrolysis of regulatory peptides, nitrogen nutrition, modulation of gene expression etc. and, consequently, are considered essential enzymes.Aminopeptidases, and more generally the proteolytic system of lactic acid bacteria (LAB) ± which are used as starters in food fermentation processes ± are essential for the nitrogen nutrition of LAB and also for the development of the organoleptic properties of dairy products (texture and flavour). Indeed, LAB are auxotrophic for several amino acids and therefore depend highly on their proteolytic system to hydrolyse milk proteins (caseins) into assimilable nitrogen compounds (small peptides and free amino acids). This proteolytic system has been studied extensively in Lactococcus lactis which is the best known species among LAB. The proteolytic cascade of casein utilization in this bacterium is now well known. This multienzymatic system is composed of a cell-wall bound proteinase, responsible for the first step of casein hydrolysis, as well as several peptide and amino-acid transport systems, and of various intracellular peptidases, mainly aminopeptidases, which are involved in further hydrolysis of the peptides produced from caseins after their transport into the cell (for...