To determine the role of the glucose gradient between the hepatoportal system (HPS) and the central nervous system (CNS) in regulating hepatic glucose uptake, experiments were conducted with seven conscious dogs using a hepatic venous catheterization technique. With the infusion of somatostatin (0.8 microg x kg(-1) x min(-1)), glucagon (0.65 ng x kg(-1) x min(-1)), and insulin (27 pmol x kg(-1) x min(-1)), arterial glucose levels could be maintained at 8 mmol/l by adjusting the intravenous glucose infusion (G(inf)) according to the following three periods: 1) peripheral glucose infusion period (PE), G(inf) alone; 2) portal glucose infusion period (PO), G(inf) plus constant glucose infusion into the portal vein (GIR(PV), 55.6 micromol x kg(-1) x min(-1)); 3) portal and brain glucose infusion period (PO+CNS), G(inf) and GIR(PV) plus additional glucose infusion into the unilateral carotid and vertebral arteries to abolish the positive glucose gradient between HPS and CNS. Arterial plasma glucose levels were clamped during the three periods (8.1 +/- 0.1, PE; 8.2 +/- 0.1, PO; 8.2 +/- 0.1 mmol/l, PO+CNS). During PO, when a positive glucose gradient was promoted between HPS and CNS, the net hepatic glucose balance (NHGB) determined by the difference between hepatic glucose inflow and outflow was significantly lower than that of PE (-41.5 +/- 5.3, PO vs. -7.5 +/- 3.4 micromol x kg(-1) x min(-1), PE; P < 0.01). However, this decrease in the NHGB significantly increased during PO+CNS, when the glucose gradient between HPS and CNS was minimized, compared with PO (-21.7 +/- 3.2 micromol x kg(-1) x min(-1), P < 0.05). We conclude that a positive glucose gradient between HPS and CNS is an important regulatory factor of hepatic glucose uptake, but other factors also play important roles because minimizing the glucose gradient between HPS and CNS diminished the net hepatic glucose uptake by 50%.