Four minor protein components were detected in whey from Romagnola cows' milk by polyacrylamide gel isoelectric focusing and two dimensional gel electrophoresis. Individual protein spots were transferred by electroblotting on to a polyvinylidene difluoride membrane and isolated by cutting out the relevant area. After in situ trypsinolysis, a portion of the digest was analysed directly by matrix-assisted laser desorption ionization–time of flight mass spectrometry. The mass profile allowed us to establish a correlation between β-lactoglobulins A and B and the four minor whey protein components. They were identified as C-terminally truncated β-lactoglobulin A and B variants with missing N-terminal peptides, beyond residues in the range 100–123 and 136–147 respectively. Two of the minor components were related to β-lactoglobulin A and two to β-lactoglobulin B.