Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of β-casein, but peptides from αs1- and αs2-caseins were also identified; the extract also contained α-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from αs1- and β- caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysis of αs2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.