1. Human erythrocyte hexokinase (ADP:D-hexose 6-phosphotransferase, EC 2.7.1.1) was purified 50 000--100 000-fold with a final specific activity of about 25--50 units/mg protein using gel-filtration, ion-exchange chromatography and affinity chromagraphy. 2. After isoelectrofocusing ofthe preparation one major protein band could be detected besides a minor band. THe isoelectric point of the major protein band was found to be 4.7. 3. After purification the enzyme could be stabilized in a medium containing inorganic phosphate, glucose, glycerol and mercaptoethanol. 4. The molecular weight was determined by gel-filtration and was found to be 132 000+/-8000. 5. The enzyme shows a broad pH optimum ranging from 7.0 to 8.4. 6. The kinetic behavior of the purified enzyme at 37 degrees C was somewhat different from the normal Michaelis-Menten kinetics due to its instability. The affinity constants were 0.048--0.080 mM for glucose and 0.57--1.0 mM for Mg-ATP. 7. The enzyme was specific for Mg- ATP as the nucleotide substrate. Mg-UTP, Mg-ITP,Mg-GTP and Mg-CTP were not converted to corresponding diphosphates. Several hexoses could be phosphorylated by the enzyme. Mannose could be phosphorylated at the same rate as glucose, although the affinity for the enzyme was lower (5m=0.60mM). Much lower rates and lower affinities were found with 2-deoxy-D-glucose (5m=1.0mM), D(+)-glucosamine (5m=4.5 mM) and fructose (5m=10 mM). N-acetyl-D-glucosamine , galactose andsorbose were not phosphorylated at all.