Glucose-induced insulin secretion is biphasic: the first phase forms a transient peak of secretion lasting a few minutes after the stimulation, whereas the second maintains a lower but persistent secretion rate. It was suggested that two different exocytic mechanisms operate during the two phases: the first phase exocytosis is derived from docked granules, whereas the second derives from newly recruited granules. However, total internal reflection fluorescence microscopy has revealed that, while the exocytosis in the first few minutes is derived from granules located close to the plasma membrane, it is also significantly effected by deeper granules, whereas that in the second phase is mainly derived from the deeper granules. Consistently, pancreatic beta cells deficient in the Rab27a effector, granuphilin, which is indispensable for the stable attachment (docking) of insulin granules to the plasma membrane, exhibit no delay or reduction of secretagogue-induced insulin secretion, and instead exhibit increased secretion. Future studies should explore the mechanism for time-dependent differences in the exocytic behaviors, namely the molecular determinants of the rate-limiting exocytic steps for docked and undocked granules.