Effects of glucagon immunoneutralization on plasma glucose, insulin, and glucagon were studied 2–4 h after intravenous injection of a high-affinity, monoclonal glucagon antibody into normal as well as moderately and severely alloxan (ALX)-induced diabetic rabbits (n = 5–7). A monoclonal trinitrophenyl antibody was used in control studies. Endogenous glucagon was completely neutralized as evidenced by undetectable levels of free glucagon and high plasma glucagon-binding capacities. In postabsorbtive normal rabbits, glucagon neutralization decreased plasma glucose by 2.2 ± 0.3 mmol/l, and the resulting plasma levels of insulin and glucagon (indirectly measured) were 8 ± 3 and 640 ± 129% of baseline, respectively. However, when euglycemia was maintained by means of glucose infusion (steady-state plasma glucose and glucose infusion rate: 6.6 ± 0.1 mmol/l and 3.0 ± 0.4 mg · kg⁻¹ · min⁻¹), both plasma insulin and glucagon remained unaltered. Thus, the glucose infusion rate accurately reflects glucagon's contribution to postabsorbtive glucose production. In both moderately and severely diabetic rabbits, immunoneutralization of glucagon decreased plasma glucose by ∼8 mmol/l, leading to euglycemia (7.3 ± 1.1 mmol/l) and reduced hyperinsulinemia (41 ± 9% of baseline) in the former and to partial restoration of euglycemia (12.7 ± 1.8 mmol/l) and unchanged insulin levels in the latter group of diabetic rabbits (P < 0.05 vs. controls in all studies). No significant changes were observed in control studies. In conclusion, glucagon is an important regulator of postabsorbtive glucose production in normal rabbits and plays an important role in the maintenance of hyperglycemia in ALX-induced diabetic rabbits.