OBJECTIVE - Given the interest in glucagon antagonism as a potential treatment of diabetes, we tested the hypothesis that glucagon, in concert with insulin, supports the postabsorptive plasma glucose concentration in humans. RESEARCH DESIGN AND METHODS - Following preliminary studies that indicated that a peripheral intravenous insulin dose of 0.1 mU · kg-1 · min-1 (lower than those used previously) provides basal insulin replacement and that a glucagon dose of 1.0 ng · kg-1 · min-1 underreplaces basal glucagon, we infused the somatostatin analog octreotide (30 ng · kg-1 · min -1) (with growth hormone replacement) over 4 h in 14 healthy adults on four separate occasions to produce endogenous insulin and glucagon deficiency with 1) saline (combined insulin and glucagon deficiency), 2) insulin replacement (isolated glucagon deficiency), 3) partial glucagon replacement (insulin and partial glucagon deficiency), and 4) insulin and partial glucagon replacement (partial glucagon deficiency). RESULTS - During combined insulin and glucagon deficiency, glucose production decreased and then increased, and mean (±SE) plasma glucose decreased from 83 ± 1 to 63 ± 2 mg/dl at 60 min and then increased to 89 ± 3 mg/dl at 240 min. During isolated glucagon deficiency, plasma glucose decreased to hypoglycemic levels and was 55 ± 2 mg/dl at 240 min (P < 0.0001 vs. combined insulin and glucagon deficiency). Partial glucagon replacement raised plasma glucose to higher levels (P = 0.0469) during insulin deficiency and to higher levels (P = 0.0090) during insulin replacement. CONCLUSIONS - These three findings provide direct evidence that glucagon, in concert with insulin, supports the postabsorptive plasma glucose concentration in humans.