Glucagon secretion from pancreatic α cells is inhibited by insulin from β cells. Amylin is a partner hormone to insulin cosecreted in response to nutrient stimuli, which, like insulin, inhibits β-cell secretion. We investigated whether amylin also inhibits α-cell secretion of glucagon in response to infused l-arginine. Rat amylin (1.2, 3.6, 12, 36, or 120 pmol/kg/min; calculated plasma concentration, 13, 47, 195, 713, and 2,950 pmol/L, respectively; n = 7, 8, 6, 4, and 7) or saline (n = 23) was infused into anesthetized male Harlan-Sprague-Dawley rats during hyperinsulinemic-euglycemic clamps, which were used to equalize the influences of glucose and insulin on glucagon secretion. Plasma glucose and insulin concentrations and mean arterial pressures were not different between amylin- and saline-treated rats during a 10-minute 2-mmol l-arginine infusion delivered during the clamps. Plasma glucagon measurements taken during and after the arginine challenge showed that compared with saline infusions, amylin administration dose-dependently suppressed the glucagon response to arginine by a maximum of 62% (incremental area under the curve [AUC] 0 to 60 minutes) with a plasma amylin EC₅₀ of 18 pmol/L ±0.3 log units. These data indicate that amylin potently inhibits arginine-stimulated glucagon secretion.