In this investigation, we sought to constrain the locus of essential portohepatic glucosensors and test the hypothesis that they reside strictly in the portal vein and not the liver. Male Wistar rats were chronically cannulated in the carotid artery (sampling), jugular vein (infusion), and portal vein, either adjacent to (POR_ADJ, 0.6 ± 0.1 cm, n = 6) or upstream from (POR_UPS, 2.7 ± 0.1 cm, n = 8) the liver. Animals were exposed to one of three protocols distinguished by the site of glucose infusion: POR_UPS, POR_ADJ, or peripheral (PER). Systemic hypoglycemia (2.4 ± 0.1 mmol/1) was induced via jugular vein insulin infusion (50 mU · kg⁻¹ · min⁻¹). Arterial plasma catecholamines were assessed at basal (−30 and 0 min) and during sustained hypoglycemia (60, 75, 90, 105 min). By design, hepatic glucose was significantly elevated during POR_UPS and POR_ADJ versus PER (4.3 ± 0.1 vs. 2.4 ± 0.1 mmol/l, respectively; P < 0.05). There were no significant differences between protocols in arterial glucose or insulin concentrations (9,372 ± 1,798 pmol/l). When liver and systemic glucose concentrations were allowed to fall concomitantly (PER), epinephrine was elevated 16-fold above basal levels (3.0 ± 0.6 vs. 46.4 ± 4.3 nmol/l, P < 0.001). When portohepatic normoglycemia was maintained during POR_UPS, a 67% suppression in the epinephrine response versus that during PER was observed (P < 0.001). However, when the cannula was advanced adjacent to the liver, by comparison with PER, there was no suppression in the sympathoadrenal response (P = 0.73). While both POR_UPS and POR_ADJ yielded elevated liver glycemia in the face of systemic hypoglycemia, only POR(_UPS yielded an elevated portal vein glucose concentration. That only POR_UPS resulted in a significant suppression of the sympathoadrenal response is consistent with the localization of the glucosensors to the portal vein.