When cells release hormones and neurotransmitters through exocytosis, cytosolic Ca(2+) triggers the fusion of secretory vesicles with the plasma membrane. It is well known that this fusion requires assembly of a SNARE protein complex. However, the timing of SNARE assembly relative to vesicle fusion essential for understanding exocytosis has not been demonstrated. To investigate this timing, we constructed a probe that detects the assembly of two plasma membrane SNAREs, SNAP25 and syntaxin-1A, through fluorescence resonance energy transfer (FRET). With two-photon imaging, we simultaneously measured FRET signals and insulin exocytosis in beta cells from the pancreatic islet of Langerhans. In some regions of the cell, we found that the SNARE complex was preassembled, which enabled rapid exocytosis. In other regions, SNARE assembly followed Ca(2+) influx, and exocytosis was slower. Thus, SNARE proteins exist in multiple stable preparatory configurations, from which Ca(2+) may trigger exocytosis through distinct mechanisms and with distinct kinetics.