Lactobacillus helveticus strains, one of the most nutritionally fastidious lactic acid bacteria, have a potent proteolytic system that makes them very interesting for different uses in the dairy industry. Its applications concern from cheese ripening to the preparation of fermented milk products with biologically active peptides. The cell-free extract (CFE) of Lactobacillus helveticus strain ITG LH1 was analysed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), using IPG immobiline dry strips (pH 4-7). With the aim to study the proteolytic enzymes expressed by Lactobacillus helveticus ITG LH1 grown in milk medium, a two step-chromatography methodology, based on ion exchange and affinity chromatography, was developed for the preparation of a peptidase-rich sub-proteome from the CFE of stationary growing cells. Several affinity chromatography columns were tested and among them a HiTrap Chelating column was selected as it provided the best performance for the enrichment in peptidases. Peptidase activities were studied using different beta-Naphtylamide (beta-NA) derivatives and specific activities were increased 50- to 100-fold by this chromatographic procedure. Sub-proteome characterisation was performed by 2D-PAGE, pH 4-7, followed by protein digestion with trypsin, analysis by MALDI-TOF mass spectrometry and subsequent database searches using peptide mass fingerprints. Among the most abundant proteins seven peptidases were present, namely the two general aminopeptidases (PepN, PepC), three dipeptidases (PepDA, PepV, PepQ) and two endopeptidases (PepO, PepO3), all of them corresponding to the catalytic classes of metallo- or cysteine-peptidases. Several stress proteins (such as heat shock proteins DnaK and GroEL) and other enzymes implied in bacterial metabolism, namely in the carbohydrate pathways (such as LDH), were also identified in the peptidase-rich sub-proteome.