In the absence of fructose 6-phosphate, the regulatory protein of rat liver glucokinase (hexokinase IV or D) inhibited this enzyme, though with a much (15-fold) lower potency than in the presence of a saturating concentration of fructose 6-phosphate. Evidence is provided that this inhibition is not due to contaminating fructose 6-phosphate. In the presence of regulatory protein, sorbitol 6-phosphate, a potent analog of fructose 6-phosphate, exerted a hyperbolic, partial inhibition on glucokinase, the degree of which increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between the rates in the absence and in the presence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] at various concentrations of regulatory protein were linear, and demonstrated that the apparent affinity for sorbitol 6-phosphate increased with the concentration of regulatory protein. Plots of the reciprocal of the difference between 1/v in the presence and in the absence of sorbitol 6-phosphate versus 1/[sorbitol 6-phosphate] were also linear and crossed the axis at a value independent of the concentration of regulatory protein. Fructose 1-phosphate released the inhibition exerted by the regulatory protein in a hyperbolic fashion. The concentration of this effector required for a half-maximal effect increased linearly with the concentrations of sorbitol 6-phosphate and of regulatory protein. These results are consistent with a model in which the regulatory protein exists under two conformations, one form which binds inhibitors and glucokinase, and the other which binds activators, although not glucokinase. Sorbitol 6-phosphate, 2-deoxysorbitol 6-phosphate and mannitol 1-phosphate, all analogs of the open-chain configuration of fructose 6-phosphate, inhibited glucokinase in the presence of regulatory protein at lower concentrations than fructose 6-phosphate, whereas fixed analogs of the furanose form of fructose 6-phosphate were inactive or behaved as activators. This indicated that fructose 6-phosphate in its open-chain configuration is recognized by the regulatory protein. A series of compounds exerted an activating effect. These included, in order of decreasing potency: fructose 1-phosphate, psicose 1-phosphate, ribitol 5-phosphate, analogs of fructose 1-phosphate and of ribitol 5-phosphate and, at much higher concentrations, inorganic phosphate.