At a concentration of 1 mM, fructose 1‐phosphate stimulated about twofold, and glucose 6‐phosphate inhibited by about 30%, the phosphorylation of 5 mM glucose in high‐speed supernatants prepared from rat liver or from isolated hepatocytes, but did not affect, or barely so, the activity of a partially purified preparation of glucokinase. Anion‐exchange chromatography of liver extracts separated glucokinase from a fructose‐6‐phosphate‐sensitive and fructose‐1‐phosphate‐sensitive inhibitor of that enzyme. This inhibitor could be further purified by chromatography on phospho‐Ultrogel. It was destroyed by trypsin and was heat‐labile. It inhibited glucokinase competitively with respect to glucose and its inhibitory effect was greatly reinforced by fructose 6‐phosphate although not by glucose 6‐phosphate. Fructose 1‐phosphate relieved the enzyme of the inhibitory effect of the regulator and antagonised the effect of fructose 6‐phosphate in a competitive manner. It is concluded that the regulator plays a role in the physiological control of the activity of glucokinase, particularly with respect to the stimulatory effect of fructose in isolated hepatocytes (see preceding paper in this journal).