The kinetic behaviour of liver glucokinase was examined in the liver cytosol from normal and streptozotocin diabetic rats. In addition to the classical reduction in glucokinase activity, diabetes severely lowered both the energy of activation and the apparent affinity of the enzyme for D-glucose. Diabetes also impaired the positive cooperativity found at low concentrations of D-glucose, whilst failing to provoke any obvious change in the anomeric specificity of the enzyme. It is proposed that such changes may contribute to the metabolic anomalies caused by diabetes mellitus in hepatocytes.