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OBJECTIVE-To characterize the voltage-gated ion channels in human ␤-cells from nondiabetic donors and their role in glucosestimulated insulin release.RESEARCH DESIGN AND METHODS-Insulin release was measured from intact islets. Whole-cell patch-clamp experiments and measurements of cell capacitance were performed on isolated ␤-cells. The ion channel complement was determined by quantitative PCR.RESULTS-Human ␤-cells express two types of voltage-gated K ϩ currents that flow through delayed rectifying (K V 2.1/2.2) and large-conductance Ca 2ϩ -activated K ϩ (BK) channels. Blockade of BK channels (using iberiotoxin) increased action potential amplitude and enhanced insulin secretion by 70%, whereas inhibition of K V 2.1/2.2 (with stromatoxin) was without stimulatory effect on electrical activity and secretion. Voltage-gated tetrodotoxin (TTX)-sensitive Na ϩ currents (Na V 1.6/1.7) contribute to the upstroke of action potentials. Inhibition of Na ϩ currents with TTX reduced glucose-stimulated (6 -20 mmol/l) insulin secretion by 55-70%. Human ␤-cells are equipped with L-(Ca V 1.3), P/Q-(Ca V 2.1), and T-(Ca V 3.2), but not N-or R-type Ca 2ϩ channels. Blockade of L-type channels abolished glucosestimulated insulin release, while inhibition of T-and P/Q-type Ca 2ϩ channels reduced glucose-induced (6 mmol/l) secretion by 60 -70%. Membrane potential recordings suggest that L-and T-type Ca 2ϩ channels participate in action potential generation. Blockade of P/Q-type Ca 2ϩ channels suppressed exocytosis (measured as an increase in cell capacitance) by Ͼ80%, whereas inhibition of L-type Ca 2ϩ channels only had a minor effect.CONCLUSIONS-Voltage-gated T-type and L-type Ca 2ϩ channels as well as Na ϩ channels participate in glucose-stimulated electrical activity and insulin secretion. Ca 2ϩ -activated BK channels are required for rapid membrane repolarization. Exocytosis of insulin-containing granules is principally triggered by Ca 2ϩ influx through P/Q-type Ca 2ϩ channels. Diabetes 57:1618-1628, 2008