Fructosamines are thought to play an important role in the development of diabetic complications. Little is known about reactions that could metabolize these compounds in mammalian tissues, except for recent indications that they can be converted to fructosamine 3-phosphates. The purpose of the present work was to identify and characterize the enzyme responsible for this conversion. Erythrocyte extracts were found to catalyze the ATP-dependent phosphorylation of 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine. The enzyme responsible for this conversion was purified approximately 2,500-fold by chromatography on Blue Sepharose, Q Sepharose, and Sephacryl S-200 and shown to copurify with a 35,000-M(r) protein. Partial sequences of tryptic peptides were derived from the protein by nanoelectrospray-ionization mass spectrometry, which allowed for the identification of the corresponding human and mouse cDNAs. Both cDNAs encode proteins of 309 amino acids, showing 89% identity with each other and homologous to proteins of unknown function predicted from the sequences of several bacterial genomes. Both proteins were expressed in Escherichia coli and purified. They were shown to catalyze the phosphorylation of DMF, fructoselysine, fructoseglycine, and fructose in order of decreasing affinity. They also phosphorylated glycated lysozyme, though not unmodified lysozyme. Nuclear magnetic resonance analysis of phosphorylated DMF and phosphorylated fructoseglycine showed that the phosphate was bound to the third carbon of the 1-deoxyfructose moiety. The physiological function of fructosamine-3-kinase may be to initiate a process leading to the deglycation of fructoselysine and of glycated proteins.