Intercellular communication is commonly mediated by the regulated fusion, or exocytosis, of vesicles with the cell surface. SNARE (soluble N-ethymaleimide sensitive factor attachment protein receptor) proteins are the catalytic core of the secretory machinery, driving vesicle and plasma membrane merger. Plasma membrane SNAREs (tSNAREs) are proposed to reside in dense clusters containing many molecules, thus providing a concentrated reservoir to promote membrane fusion. However, biophysical experiments suggest that a small number of SNAREs are sufficient to drive a single fusion event. Here we show, using molecular imaging, that the majority of tSNARE molecules are spatially separated from secretory vesicles. Furthermore, the motilities of the individual tSNAREs are constrained in membrane micro-domains, maintaining a non-random molecular distribution and limiting the maximum number of molecules encountered by secretory vesicles. Together our results provide a new model for the molecular mechanism of regulated exocytosis and demonstrate the exquisite organization of the plasma membrane at the level of individual molecular machines.