Insulin is secreted from pancreatic β cells in response to an elevation of cytoplasmic Ca 2+ resulting from enhanced Ca 2+ influx through voltage‐gated Ca 2+ channels. Mouse β cells express several types of Ca 2+ channel (L‐, R‐and possibly P/Q‐type). β cell‐selective ablation of the gene encoding the L‐type Ca 2+ channel subtype Ca v 1.2 (βCa v 1.2−/− mouse) decreased the whole‐cell Ca 2+ current by only∼ 45%, but almost abolished first‐phase insulin secretion and resulted in systemic glucose intolerance. These effects did not correlate with any major effects on intracellular Ca 2+ handling and glucose‐induced electrical activity. However, high‐resolution capacitance measurements of exocytosis in single β cells revealed that the loss of first‐phase insulin secretion in the βCa v 1.2−/− mouse was associated with the disappearance of a rapid component of exocytosis reflecting fusion of secretory granules physically attached to the Ca v 1.2 channel. Thus, the conduit of Ca 2+ entry determines the ability of the cation to elicit secretion.