Glucokinase reversibly partitions between a bound and a free state in the hepatocyte in response to the metabolic status of the cell. Maximum binding occurs at low [glucose] (<5 mM) and minimum binding at high [glucose] or in the presence of sorbitol or fructose. In this study we determined the binding characteristics of glucokinase in the hepatocyte in situ, by adenovirus-mediated glucokinase overexpression combined with the digitonin-permeabilization technique. We also determined the sensitivity of glycogen synthesis to changes in either total glucokinase overexpression or in free glucokinase activity. Glucokinase overexpression is associated with an increase in both free and bound activity, with an overall decrease in the proportion of bound activity. In hepatocytes incubated at low [glucose] (0-5 mM), glucokinase binding involves a high-affinity binding site with a Kd of approximately 0.1 microM and a binding capacity of approximately 3 pmol/mg total cell protein and low-affinity binding with a Kd of approximately 1.6 microM. Increasing glucose concentration to 20 mM causes a dose-dependent increase in the Kd of the high- affinity site to approximately 0.6 microM, and this effect was mimicked by 50 microM sorbitol, a precursor of fructose 1-P, confirming that this site is the regulatory protein of glucokinase. Glycogen synthesis determined from the incorporation of [2-3H,U-14C]glucose into glycogen at 5 mM or 10 mM glucose was very sensitive to small increases in total glucokinase activity and correlated more closely with the increase in free glucokinase activity. The relation between glycogenic flux and glucokinase activity is sigmoidal. Expression of the sensitivity of glycogen synthesis to glucokinase activity as the control coefficient reveals that the coefficient is greater for the incorporation of 2-tritium (which occurs exclusively by the direct pathway) than for incorporation of 14C label (which involves direct and indirect pathways) and is greater at 5 mM glucose (when glucokinase is maximally sequestered at its high-affinity site) than at 10 mM glucose. The results support the hypothesis that compartmentation of glucokinase in the hepatocyte increases the sensitivity of glycogen synthesis to small changes in total glucokinase activity and that glucose-induced translocation of glucokinase has a major role in the acute control of glycogen synthesis.