The number and nature of glucose-phosphorylating enzymes of rat intestinal mucosa were investigated by chromatographic, electrophoretic, and kinetic methods. Three fractions with glucose-phosphorylating activity were obtained from the supernatant fluid of mucosa homogenate by means of DEAE-cellulose chromatography, corresponding to hexokinases A and B (EC 2.7.1.1.), and N-acetyl-D-glucosamine kinase (EC 2.7.1.59). Although the latter uses N-acetylglucosamine as the main substrate, it is also able to phosphorylate glucose. Electrophoresis in polyacrylamide and in cellulose acetate gels showed the same three enzyme activities. None of these procedures revealed the presence of either hexokinase D ("glucokinase") or hexokinase C in the intestinal mucosa. In the sediment fractions hexokinase A and B, but not N-acetylglucosamine kinase, were found. The Km values for glucose of partially purified hexokinases A and B were 0.025 and 0.174 mM, respectively, and their substrate specificity was the same as that of hexokinases A or B from other tissues. Partially purified N-acetylglucosamine kinase showed hyperbolic saturation functions for N-acetylglucosamine and ATP, with Km values of 0.021 and 0.38 mM, respectively. This enzyme also phosphorylated glucose, mannose, fructose, 2-deoxyglucose, and glucosamine. The dependence of velocity on glucose concentrations was complex, mimicking negative cooperativity. The molecular weight of both hexokinases A and B was 98,000 and that of N-acetylglucosamine kinase was 59,000. The kinetic properties, as well as the chromatographic and electrophoretic mobilities, of N-acetylglucosamine kinase may serve to confuse it with hexokinase D, and thus several criteria should be applied for correct identification.