N-Acetylglucosamine, a major sugar in complex carbohydrates, enters the pathways of aminosugar metabolism by the action of N-acetylglucosamine kinase (EC 2.7.1.59). In this study we report the purification to homogeneity of GlcNAc kinase from rat liver cytosol using salmine sulfate precipitation, chromatography on phenyl-Sepharose, ATP-agarose and MonoQ, and finally gel filtration on Superdex 200. It was characterized as a dimer of 39-kDa subunits. About 25% of the amino acid sequence of GlcNAc kinase was established by peptide mapping. Part of the ATP-binding site of GlcNAc kinase was identified by sequence comparison with related hexokinases, including the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase (EC 5.1.3.14/2.7.1.60), the key enzyme of N-acetylneuraminic acid biosynthesis in rat liver. The Cys residues in the active sites of GlcNAc kinase and ManNAc kinase were characterized by chemical modification with N-ethylmaleimide, iodoacetamide and 5,5'-dithiobis(2-nitrobenzoic acid). The finding that the substrates GlcNAc and ManNAc protected their respective enzymes from inhibition by the above sulfhydryl reagents indicates that Cys residues are located in or near the active sites of both enzymes. Use of the specific dithiol-modifying chemical reagents, sodium meta-periodate, sodium meta-arsenite/2,3-dimercaptopropanol and diazenedicarboxylic acid bis-N,N'-dimethylamide revealed that the active sites of GlcNAc kinase and ManNAc kinase possess at least one pair of vicinal thiols. Chemical treatment of UDP-GlcNAc 2-epimerase provided no evidence for the presence of cysteine in the active site of this enzyme. From the incorporation of N-[3H]ethylmaleimide into GlcNAc kinase in the absence and presence of ligands we estimated that the active site of GlcNAc kinase contains two Cys residues.