To determine the role of nitric oxide in regulating net hepatic glucose uptake (NHGU) in vivo, studies were performed on three groups of 42-h-fasted conscious dogs, using a nitric oxide donor (SIN-1). The experimental period was divided into P1 (0-90 min) and P2 (90-240 min). At 0 min, somatostatin was infused peripherally and insulin (4-fold basal) and glucagon (basal) were given intraportally. Glucose was delivered intraportally (22.2 µmol/kg/min) and peripherally (as needed) to increase the hepatic glucose load 2-fold basal. At 90 min, an infusion of SIN-1 (4 µg/kg/min) was started into a peripheral vein (PeSin-1, n=10) or the portal vein (PoSin-1, n=12), while the control group received saline (SAL, n=8). Both peripheral and portal infusion of SIN-1, unlike saline, significantly reduced systolic and diastolic blood pressure. Heart rate rose in PeSin-1 and PoSin-1 (96±5 to 120±10 and 88±6 to 107±5 bpm, respectively, P<0.05), but did not change in response to saline. NHGU during P2 was 31.0±2.4 and 29.9±2.0 µmol/kg/min in SAL and PeSin-1, respectively, but was 23.7±1.7 in PoSin-1 (P<0.05). Net hepatic carbon retention during P2 was significantly lower in PoSin-1 than SAL or PeSin-1 (21.4±1.2 vs. 27.1±1.5 and 26.1±1.0 µmol/kg/min). Nonhepatic glucose uptake did not change in response to saline or SIN-1 infusion. In conclusion, portal but not peripheral infusion of the nitric oxide donor SIN-1 inhibited NHGU.