The fructose-6-phosphate-sensitive and fructose-1-phosphate-sensitive protein that inhibits rat liver glucokinase [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179-184] was purified close to homogeneity by a procedure involving poly(ethyleneglycol) precipitation, chromatography on anion-exchangers and hydroxylapatite, gel filtration and chromatography on Mono S, a cation exchanger. In the last chromatographic step, the regulatory protein coeluted with a 62 kDa peptide. From the elution volume of the gel-filtration column a molecular mass of 60 kDa was determined, allowing the conclusion that the regulator is a monomer. The decrease in the apparent affinity of glucokinase for glucose, which the regulator induced, disappeared upon separation of the two proteins. Furthermore, the regulator did not appear to catalyse the formation of a heat-stable or a trypsin-resistant inhibitor of glucokinase. Finally, the inhibition exerted by the regulatory protein reached a steady value 1-2 min after the addition of the regulator. These results indicate that the regulator does not act by causing a covalent modification of glucokinase nor by catalysing the formation of a low-molecular-mass inhibitor. Raising the concentration of glucokinase in the assay from 6 mU/ml to 120 mU/ml caused a 2.5-fold increase in the concentration of regulator required to half-maximally inhibit the enzyme. The apparent mass of glucokinase, as determined by centrifugation in isokinetic sucrose gradient, was 55 kDa, and this value was unaffected by the separate presence of fructose 6-phosphate or of the regulatory protein. However, the apparent mass of the enzyme increased to 105 kDa when glucokinase was centrifuged together with both fructose 6-phosphate and the regulatory protein, although not when fructose 1-phosphate was also present. Conversely, the presence of glucokinase increased the apparent molecular mass of the regulator in the presence of fructose 6-phosphate. From these results, it is concluded that the regulatory protein inhibits glucokinase by forming a complex with this enzyme in the presence of fructose 6-phosphate, and that fructose 1-phosphate antagonises this inhibition by preventing the formation of the complex.