Using a binding assay in which the ligand-protein complex is separated from free ligand by precipitation with poly(ethylene glycol) 6000, we found that the regulatory protein of rat liver glucokinase bound close to 1 mol of radiolabelled sorbitol 6-phosphate, a negative effector, or of fructose 1-phosphate, a positive effector, per mol of regulatory protein. Scatchard plots were linear, the dissociation constant being 0.3 microM for both phosphate esters. Sorbitol 6-phosphate and fructose 1-phosphate competed with each other for the binding. Competition was also observed with psicose 1-phosphate, ribitol 5-phosphate, arabitol 5-phosphate and 3-phosphoglycerate, all of which are known to affect the inhibition exerted by the regulatory protein. At a concentration of 10%, poly(ethylene glycol) 6000 decreased the concentration of regulatory protein causing 50% inhibition to a larger extent in the absence (12-fold) than in the presence (3-fold) of a saturating concentration of fructose 6-phosphate, another negative effector. Furthermore, it increased by about 3-fold the apparent affinity for inhibitory phosphate esters, indicating that it induced conformational changes of the regulatory protein.