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 [3-morpholinosydnonimine (SIN-1)]. The experimental period was divided into period 1 (0-90 min) and period 2 (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 mumol.kg(-1).min(-1)) and peripherally (as needed) to increase the hepatic glucose load twofold basal. At 90 min, an infusion of SIN-1 (4 mug.kg(-1).min(-1)) was started in 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 beats/min, 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 mumol.kg(-1).min(-1) 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 mumol.kg(-1).min(-1)). 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.