1. 1. Human hexokinase (HK) I was partially purified from muscle and liver, HK II from hepatoma and HK III from liver. 2. 2. Another HK isoenzyme with electrophoretic mobility intermediate between that of HK II and HK III, and staining more heavily with high concentrations of glucose than with low concentrations, was first observed in human foetal tissues, and partially purified from foetal brain (HK II f). 3. 3. The Michaelis constants for MgATP were similar for all the isoenzymes tested, while those for glucose differed, with that for HK III < HK I < HK II f < HK II. The Michaelis constant was equal to the dissociation constant for all the isoenzymes tested except liver HK I and hepatoma HK II. 4. 4. The only isoenzyme which was inhibited by high glucose levels was adult liver hexokinase III. 5. 5. All the hexokinase isoenzymes were found to follow a sequential mechanism of substrate addition and to have a higher affinity for glucose than for fructose.