When tested in the presence of an inhibitor of sorbitol dehydrogenase, both mannitol and sorbitol caused a progressive inhibition of the detritiation of [2-3H]glucose in isolated rat hepatocytes. The purpose of the present work was to investigate the possibility that this effect was mediated by the regulatory protein of glucokinase. When added to hepatocytes, mannitol decreased the apparent affinity of glucokinase for glucose and increased the concentration of fructose required to stimulate detritiation, without affecting the concentration of fructose 1-phosphate. Its effect could be attributed to the formation of mannitol 1-phosphate, a potent agonist of the regulatory protein, which, similarly to fructose 6-phosphate, reinforces its inhibitory action. Formation of mannitol 1-phosphate in hepatocytes was dependent on the presence of mannitol and was stimulated by compounds that increase the concentration of glucose 6-phosphate. Liver extracts catalysed the conversion of mannitol to mannitol 1-phosphate about 7 times more rapidly in the presence of glucose 6-phosphate than of ATP. The glucose 6-phosphate-dependent formation was entirely accounted for by a microsomal enzyme, glucose-6-phosphatase and was not due to a loss of latency of this enzyme. In hepatocytes in primary culture, mannitol decreased the detritiation rate and counteracted the effect of fructose to stimulate glucokinase translocation. Taken together, these results strongly support a central role played by the regulatory protein in the control of glucokinase activity and translocation in the liver, as well as a feedback control exerted by fructose 6-phosphate on this enzyme.