Fructosamine-3-kinase (FN3K) phosphorylates fructosamine residues, leading to their destabilization and their shedding from protein. Support for the occurrence of this deglycation mechanism in intact cells has been obtained by showing that hemoglobin is significantly more glycated when human erythrocytes are incubated with an elevated glucose concentration in the presence of 1-deoxy-1-morpholinofructose (DMF), a cell-permeable inhibitor of FN3K, than in its absence. The aim of this work was to identify the fructosamine residues on hemoglobin that are removed as a result of the action of FN3K in intact erythrocytes. Highly glycated hemoglobin derived from intact human erythrocytes incubated for 48 h with 200 mm glucose and DMF was incubated in vitro with FN3K and [gamma-(32)P]ATP. After reduction of fructosamine 3-phosphates with borohydride, the protein was digested with trypsin. Peptides were separated by reversed-phase high-performance liquid chromatography, and the radioactive peaks were analyzed by mass spectrometry. Nine different modified residues were identified. These were Lys-alpha-16, Lys-alpha-61, Lys-alpha-139, Val-beta-1, Lys-beta-17, Lys-beta-59, Lys-beta-66, Lys-beta-132, and Lys-beta-144. Some (e.g. Lys-alpha-139) were readily phosphorylated to a maximal extent by FN3K in vitro whereas others (e.g. Val-beta-1) were slowly and only very partially phosphorylated. The radiolabeled peptides containing reduced fructosamine 3-phosphates bound to Lys-alpha-16, Lys-alpha-139, and Lys-beta-17 were much less abundant if the hemoglobin substrate used for the in vitro phosphorylation with FN3K and [gamma-(32)P]ATP came from erythrocytes incubated with an elevated glucose concentration in the absence of DMF, indicating that these lysine residues had been substantially deglycated in intact cells when FN3K action was unrefrained. Other residues (e.g. Val-beta-1, Lys-alpha-61) seemed to be insignificantly deglycated in intact cells.