The water-soluble extract of Cheddar cheese was fractionated by diafiltration using 10 kDa cut-off membranes. Peptides were isolated from the diafiltrate retentate by chromatography on DEAE-cellulose with a linear NaCl gradient in 50 mM-Tris-HCl. pH 8.6, and reversed-phase HPLC or electroblotting from urea-PAGE gels. Peptides were identified by determining N-terminal amino acid sequences and mass spectrometry. Most (45) of the total 51 peptides identified in the diafiltrate retentate originated from beta-casein, especially from a short region in the N-terminal half of the molecule. Only six peptides originated from alpha s1-casein; beta-lactoglobulin was also identified in the retentate. The origin of most of these peptides could be explained on the basis of known specificities of lactococcal cell envelope proteinases.