Glucose phosphorylating activity of human erythrocytes quickly decreases during cell ageing; the electrophoretic pattern suggests that this fast decrease is due mainly to the isozyme II. We have shown that in the young cells only hexokinase I and II are responsible for the glucose phosphorylation, while in the old cells another glucose phosphorylating activity, more evident at high glucose concentration, is also present. The appearance of this activity during cell ageing could be interpreted as a post-translational modification of the native hexokinase.