To study effects of the portal-arterial glucose difference on the hepatic glycogenesis, the liver was isolated from fasted rats and was bivascularly perfused. Thirty-five milliliters of Krebs-Ringer buffer (pH 7.4) with 2 mM glucose, 3 mM lactate, 20 ng/ml insulin, and [1-14C]glucose or [U-14C]lactate was recirculated at flow rates of 14 ml/min via the portal vein and 7 ml/min via the hepatic artery. Glucose was continuously infused at a rate of 27.75 mumol/min into the portal (P experiment) and the arterial cannula (A experiment), and the portal-arterial glucose gradients were +1.98 and -3.96 mM. Perfusate glucose concentration was not different between the P and A experiments within 20 min. Perfusate lactate level was higher in the P experiment than in the A experiment at 20 min. Incorporation of radioactivity from [14C]glucose into glycogen was higher in the P experiment than in the A experiment (0.245 +/- 0.014%/20 min vs 0.175 +/- 0.022%/20 min, P < 0.01), and not influenced by the addition of insulin. Incorporation of 14C from [14C]lactate into glycogen was not different between the P and A experiments, and was significantly increased with the addition of insulin. This activity, in the presence of insulin, was higher in the P experiment than in the A experiment (0.490 +/- 0.028%/20 min vs 0.406 +/- 0.025%/20 min, P < 0.05). These results suggest that the portal-arterial glucose difference has an important role in the regulation of hepatic glycogenesis from exogenous glucose and gluconeogenesis.