We previously reported that simulation of the chronic hyperglucagonemia seen during infection was unable to recreate the infection-induced increase in hepatic glucose production. However, chronic hyperglucagonemia was accompanied by a fall in the arterial levels of gluconeogenic precursors as opposed to a rise as is seen during infection. Thus our aim was to determine whether an infusion of gluconeogenic precursors could increase hepatic glucose production in a setting of hyperglucagonemia. Studies were done in 11 conscious chronically catheterized dogs in which sampling (artery and portal and hepatic veins) and infusion catheters (splenic vein) were implanted 17 days before study. Forty-eight hours before infusion of gluconeogenic (GNG) precursors, a sterile fibrinogen clot was placed into the peritoneal cavity. Glucagon was infused over the subsequent 48-h period to simulate the increased glucagon levels (~500 pg/ml) seen during infection. On the day of the experiment, somatostatin was infused peripherally, and basal insulin and simulated glucagon were infused intraportally. After a basal period, a two-step increase in lactate and alanine was initiated (120 min/step; n = 5). Lactate (479 ± 25 and 1,780 ± 85 µM; expressed as change from basal in periods I and II, respectively) and alanine (94 ± 13 and 287 ± 44 µM) levels were increased. Despite increases in net hepatic GNG precursor uptake (0.7 ± 0.3 and 1.1 ± 0.4 mg glucose · kg¹ · min¹), net hepatic glucose output did not increase. Because nonesterified fatty acid (NEFA) levels fell, in a second series of studies, the fall in NEFA was eliminated. Intralipid and heparin were infused during the two-step substrate infusion to maintain the NEFA levels constant in period I and increase NEFA availability in period II ( 29 ± 29 and 689 ± 186 µM; n = 6). In the presence of similar increases in net hepatic GNG precursor uptake and despite increases in arterial glucose levels (17 ± 5 and 38 ± 12 mg/dl), net hepatic glucose output increased (0.6 ± 0.1 and 0.7 ± 0.2 mg · kg¹ · min¹). In summary, a chronic increase in glucagon, when combined with an acute increase in gluconeogenic precursor and maintenance of NEFA supply, increases hepatic glucose output as is seen during infection.