N-Acetylglucosamine is a major component of complex carbohydrates. The mammalian salvage pathway of N-acetylglucosamine recruitment from glycoconjugate degradation or nutritional sources starts with phosphorylation by N-acetylglucosamine kinase. In this study we describe the identification of two active site cysteines of the sugar kinase by site-directed mutagenesis and computer-based structure prediction. Murine N-acetylglucosamine kinase contains six cysteine residues, all of which were mutated to serine residues. The strongest reduction of enzyme activity was found for the mutant C131S, followed by C143S. Determination of the kinetic properties of the cysteine mutants showed that the decreased enzyme activities were due to a strongly decreased affinity to either N-acetylglucosamine for C131S, or ATP for C143S. A secondary structure prediction of N-acetylglucosamine kinase showed a high homology to glucokinase. A model of the three-dimensional structure of N-acetylglucosamine kinase based on the known structure of glucokinase was therefore generated. This model confirmed that both cysteines are located in the active site of N-acetylglucosamine kinase with a potential role in the binding of the transferred gamma-phosphate group of ATP within the catalytic mechanism.