Antecedent hypoglycemia leads to impaired counterregulation and hypoglycemic unawareness. To ascertain whether antecedent hypoglycemia impairs portal vein glucose sensing, thereby inducing counterregulatory failure, we compared the effects of antecedent hypoglycemia, with and without normalization of portal vein glycemia, upon the counterregulatory response to subsequent hypoglycemia. Male rats were chronically cannulated in the carotid artery (sampling), jugular vein (glucose and insulin infusion), and mesenteric vein (glucose infusion). On day 1, 3 distinct antecedent protocols were employed: 1) HYPO-HYPO: systemic hypoglycemia (2.52±0.11 mM); 2) HYPO-EUG: systemic hypoglycemia (2.70 ± 0.03 mM) with normalization of portal vein glycemia (portal vein glucose = 5.86±0.10 mM); 3) EUG-EUG: systemic euglycemia (6.33 ± 0.31 mM). On day 2, all groups underwent a hyperinsulinimic-hypoglycemic clamp with the glycemic nadir (2.34 ± 0.04 mM) reached by mn 75. Hormonal responses were measured at basal and hypoglycemia (60-105 min). Compared to EUG-EUG, antecedent hypoglycemia (HYPO-HYPO) significantly blunted the peak epinephrine (10.44±1.35 vs. 15.75±1.33 nM: p=0.01) and glucagon (341±16 vs. 597±82 pg/ml: p=0.03) responses to next day hypoglycemia. Normalization of portal glycemia during systemic hypoglycemia on day 1 (HYPO-EUG) prevented blunting of the peak epinephrine (15.59±1.43 vs. 15.75±1.33 nM: p=0.94) and glucagon (523±169 vs. 597±82 pg/ml: p=0.66) responses to day 2 hypoglycemia. Consistent with hormonal responses, the glucose infusion rate during day 2 hypoglycemia was substantially elevated in HYPO-HYPO (74±12 vs. 49±4 µmol·kg^-1·min^-1; P=0.03) but not HYPO-EUG (39±7 vs. 49±4 µmol·kg^-1·min^-1: p=0.36). Antecedent hypoglycemia local to the portal vein is required for the full induction of HAAF with slow-onset hypoglycemia.