Glucokinase, an enzyme that catalyzes the phosphorylation of glucose, constitutes the key regulatory step in glucose metabolism in pancreatic islets and liver. We found that 3-O-methyl-N-acetyl-D-glucosamine (3-O-methyl-GlcNAc) potently inhibits glucose phosphorylation by N-acetylglucosamine kinase whereas glucokinase is not at all affected by this hexosamine. The addition of 3-O-methyl-GlcNAc to the assay system for glucokinase in rat liver extracts, which contain a high activity of glucokinase (glucose as substrate) relative to N-acetylglucosamine kinase (N-acetyl-D-glucosamine as substrate), affected neither Km nor Vmax values of glucokinase. On the other hand, both Km and Vmax values of glucokinase in rat pancreatic islet extracts, in which N-acetylglucosamine kinase activity is higher than glucokinase activity, were significantly lowered by the use of 3-O-methyl-GlcNAc as an inhibitor of N-acetylglucosamine kinase.