In hepatocytes glucokinase (GK) and glucose-6-phosphatase (Glc-6-Pase)1 have converse effects on glucose 6-phosphate (and fructose 6-phosphate) levels. To establish whether hexose 6-phosphate regulates GK binding to its regulatory protein, we determined the effects of Glc-6-Pase overexpression on glucose metabolism and GK compartmentation. Glc-6-Pase overexpression (4- fold) decreased glucose 6-phosphate levels by 50% and inhibited glycogen synthesis and glycolysis with a greater negative control coefficient on glycogen synthesis than on glycolysis, but it did not affect the response coefficients of glycogen synthesis or glycolysis to glucose, and it did not increase the control coefficient of GK or cause dissociation of GK from its regulatory protein, indicating that in hepatocytes fructose 6-phosphate does not regulate GK translocation by feedback inhibition. GK overexpression increases glycolysis and glycogen synthesis with a greater control coefficient on glycogen synthesis than on glycolysis. On the basis of the similar relative control coefficients of GK and Glc-6-Pase on glycogen synthesis compared with glycolysis, and the lack of effect of Glc-6-Pase overexpression on GK translocation or the control coefficient of GK, it is concluded that the main regulatory function of Glc-6-Pase is to buffer the glucose 6-phosphate concentration. This is consistent with recent findings that hyperglycemia stimulates Glc-6-Pase gene transcription.