Fructose activates glucokinase by releasing the enzyme from its inhibitory protein in liver. To examine the importance of acute activation of glucokinase in regulating hepatic glucose uptake, the effect of intraportal infusion of a small amount of fructose on net hepatic glucose uptake (NHGU) was examined in 42 h-fasted conscious dogs. Isotopic ([3-3H] and [U-14C]glucose) and arteriovenous difference methods were used. Each study consisted of an equilibration period (-90 to -30 min), a control period (-30 to 0 min), and a hyperglycemic/hyperinsulinemic period (0-390 min). During the latter period, somatostatin (489 pmol x kg(-1) x min(-1)) was given, along with intraportal insulin (7.2 pmol x kg(-1) x min(-1)) and glucagon (0.5 ng x kg(-1) x min(-1)). In this way, the liver sinusoidal insulin level was fixed at four times basal (456 +/- 60 pmol/l), and liver sinusoidal glucagon level was kept basal (46 +/- 6 ng/l). Glucose was infused through a peripheral vein to create hyperglycemia (12.5 mmol/l plasma). Hyperglycemic hyperinsulinemia (no fructose) switched net hepatic glucose balance (micromoles per kilogram per minute) from output (11.3 +/- 1.4) to uptake (14.7 +/- 1.7) and net lactate balance (micromoles per kilogram per minute) from uptake (6.5 +/- 2.1) to output (4.4 +/- 1.5). Fructose was infused intraportally at a rate of 1.7, 3.3, or 6.7 micromol x kg(-1) x min(-1), starting at 120, 210, or 300 min, respectively. In the three periods, portal blood fructose increased from <6 to 113 +/- 14, 209 +/- 29, and 426 +/- 62 micromol/l, and net hepatic fructose uptake increased from 0.03 +/- 0.01 to 1.3 +/- 0.4, 2.3 +/- 0.7, and 5.1 +/- 0.6 micromol x kg(-1) x min(-1), respectively. NHGU increased to 41 +/- 3, 54 +/- 5, and 69 +/- 8 micromol x kg(-1) x min(-1), respectively, and net hepatic lactate output increased to 11.0 +/- 3.2, 15.3 +/- 2.7, and 22.4 +/- 2.8 micromol x kg(-1) x min(-1) in the three fructose periods, respectively. The amount of [3H]glucose incorporated into glycogen was equivalent to 69 +/- 3% of [3H]glucose taken up by the liver. These data suggest that glucokinase translocation within the hepatocyte is a major determinant of hepatic glucose uptake by the dog in vivo.