The extent of the early stage of the Maillard-type reaction that impaired functional properties of whey proteins was evaluated by electrospray ionization mass spectrometry. Under conditions of mild heat treatment (63 degrees C for 20 s) applied to milk before whey separation at room temperature 23 degrees C), a modification of the relative molecular mass of beta-lactoglobulin (beta-LG) was observed that differed from that of the native form by 324. This specific modification of beta-LG occurred in acidified whey as well as in sweet whey and increased with the extent of the heat treatment. Incubation of purified beta-LG dissolved in milk ultrafiltration permeate or in lactose solution at 50 to 80 degrees C demonstrated the presence of a lactosyl residue that was covalently bound to beta-LG; beta-casein, used as a control, showed no mass modification. Studies of kinetics showed that a maximum of 35% of the beta-LG was lactosyl-beta-LG conjugate after heat treatment at 70 degrees C for 1 h. This study provides the first direct evidence of specific lactosylation of beta-LG during the initial stage of the Maillard reaction. One of the first lactose-binding sites was identified as a Lys47 by protease mapping and analysis by means of on-line liquid chromatography combined with mass spectrometry. In addition, collision-activated dissociation performed on the lactosylated peptide beta-LG (f 46-51) showed the rearrangement reactions occurring during the fragmentation process by electrospray. A mechanism is proposed.