In isolated rat pancreatic α-cells, glucose, arginine, and the sulfonylurea tolbutamide stimulated glucagon release. The effect of glucose was abolished by the K_ATP-channel opener diazoxide as well as by mannoheptulose and azide, inhibitors of glycolysis and mitochondrial metabolism. Glucose inhibited K_ATP-channel activity by 30% (P < 0.05; n = 5) and doubled the free cytoplasmic Ca²⁺ concentration. In cell-attached recordings, azide opened K_ATP channels. The N-type Ca²⁺-channel blocker ω-conotoxin and the Na⁺-channel blocker tetrodotoxin inhibited glucose-induced glucagon release whereas tetraethylammonium, a blocker of delayed rectifying K⁺ channels, increased secretion. Glucagon release increased monotonically with increasing K⁺ concentrations. ω-Conotoxin suppressed glucagon release to 15 mm K⁺, whereas a combination of ω-conotoxin and an L-type Ca²⁺-channel inhibitor was required to abrogate secretion in 50 mm K⁺. Recordings of cell capacitance revealed that glucose increased the exocytotic response evoked by membrane depolarization 3-fold. This correlated with a doubling of glucagon secretion by glucose in intact rat islets exposed to diazoxide and high K⁺. In whole-cell experiments, exocytosis was stimulated by reducing the cytoplasmic ADP concentration, whereas changes of the ATP concentration in the physiological range had little effect. We conclude that glucose stimulates glucagon release from isolated rat α-cells by K_ATP-channel closure and stimulation of Ca²⁺ influx through N-type Ca²⁺ channels. Glucose also stimulated exocytosis by an amplifying mechanism, probably involving changes in adenine nucleotides. The stimulatory action of glucose in isolated α-cells contrasts with the suppressive effect of the sugar in intact islets and highlights the primary importance of islet paracrine signaling in the regulation of glucagon release.