Pancreatic α-cells, like β-cells, express ATP-sensitive K⁺ (K_ATP) channels. To determine the physiological role of K_ATP channels in α-cells, we examined glucagon secretion in mice lacking the type 1 sulfonylurea receptor (Sur1). Plasma glucagon levels, which were increased in wild-type mice after an overnight fast, did not change in Sur1 null mice. Pancreas perfusion studies showed that Sur1 null pancreata lacked glucagon secretory responses to hypoglycemia and to synergistic stimulation by arginine. Pancreatic α-cells isolated from wild-type animals exhibited oscillations of intracellular free Ca²⁺ concentration ([Ca²⁺]_i) in the absence of glucose that became quiescent when the glucose concentration was increased. In contrast, Sur1 null α-cells showed continuous oscillations in [Ca²⁺]_i regardless of the glucose concentration. These findings indicate that K_ATP channels in α-cells play a key role in regulating glucagon secretion, thereby adding to the paradox of how mice that lack K_ATP channels maintain euglycemia.