The relative importance of insulin and glucagon as primary regulators of glucose metabolism in vivo was assessed in 18-hour fasted conscious dogs. Glucose turnover was determined using [3-³H]glucose and gluconeogenesis was assessed using tracer ([¹⁴C]alanine) and A-V difference techniques during a 40-minute control period and a 3-hour period during which various hormonal perturbations were brought about. During the infusion of somatostatin and basal intraportal replacement amounts of insulin and glucagon for the entire study, the plasma glucose concentration (109 ± 5 mg/dL), glucose production (3.24 ± 0.30 mg/kg/min), and glucose utilization (3.17 ± 0.32 mg/kg/min) remained unchanged. When the glucagon infusion rate was increased fourfold at the end of the control period, the plasma glucose level increased from 107 ± 4 to 225 ± 23 mg/dL by 1 hour and remained elevated. Glucose production increased from 3.14 ± 0.29 to 7.66 ± 0.51 mg/kg/min by 15 minutes and decreased to 4.23 ± 0.35 mg/kg/min by 3 hours. Glucose utilization rose from a basal value of 3.20 ± 0.26 to 5.46 ± 0.27 mg/kg/min by 3 hours. When a fourfold increase in the insulin infusion rate was brought about at the end of the control period, glucose production decreased from 2.83 ± 0.20 to 1.16 ± 0.57 mg/kg/min by 1 hour, after which it increased slightly (1.62 ± 0.81 mg/kg/min). Glucose utilization increased from 2.92 ± 0.30 to 8.12 ± 1.12 mg/kg/min by 3 hours. Euglycemia was maintained by glucose infusion. Concomitant fourfold increases in the insulin and glucagon infusion rates caused glucose production to fall from 3.03 ± 0.23 to 0.76 ± 0.39 mg/kg/min by 1 hour and to remain similarly suppressed for 3 hours. Glucose utilization rose from 2.95 ± 0.20 to 8.44 ± 0.87 mg/kg/min and euglycemia was again maintained by glucose infusion. Gluconeogenic conversion increased (67 ± 12%) in the studies in which insulin and glucagon were kept at basal values, but the level, fractional extraction, and hepatic uptake of alanine did not change significantly. The selective increase in glucagon caused gluconeogenic conversion (169% ± 42%), hepatic fractional alanine extraction (0.32 ± 0.05 to 0.66 ± 0.10), and hepatic alanine uptake (2.96 ± 0.45 to 4.54 ± 0.43 μmol/kg/min) to increase, while the alanine level decreased (387 ± 40 to 272 ± 48 μmol/L). The selective increase in insulin was associated with an increase in gluconeogenic conversion (40% ± 25%) similar to that apparent in the control group, no change in the fractional extraction or uptake of alanine by the liver, and a small fall in alanine level (337 ± 33 to 249 ± 55 μmol/L). Concomitant increases in both insulin and glucagon left gluconeogenic conversion unchanged (26% ± 21%), but increased the fractional extraction of alanine by the liver (0.39 ± 0.05 to 0.64 ± 0.03). Hepatic alanine uptake did not change, but a decrease in the alanine level (353 ± 40 to 174 ± 16 μmol/L) was observed. These studies indicate that in the overnight fasted conscious dog, insulin is a potent inhibitor of the stimulatory effects of glucagon on hepatic glycogenolysis and gluconeogenesis. However, insulin is unable to limit alanine uptake by suppression of hepatic fractional extraction, instead it prevents increased alanine uptake by limiting the net release of alanine from extrahepatic tissues.