Lipid microdomains can selectively include or exclude proteins and may be important in a variety of functions such as protein sorting, cell signaling, and synaptic transmission. The present study demonstrates that two different voltage-gated calcium channels, which both interact with soluble N-ethyl-maleimide-sensitive fusion protein attachment protein receptor (SNARE) proteins but have distinct subcellular distributions and roles in synaptic transmission, are differently distributed in lipid microdomains; presynaptic P/Q (Cav2.1) but not Lc (Cav1.2) calcium channel subtypes are mainly accumulated in detergent-insoluble complexes. The immunoisolation of multiprotein complexes from detergent-insoluble or detergent-soluble fractions shows that the alpha1A subunits of Cav2.1 colocalize and interact with SNARE complexes in lipid microdomains. The altered organization of these microdomains caused by saponin and methyl-beta-cyclodextrin treatment largely impairs the buoyancy and distribution of Cav2.1 channels and SNAREs in flotation gradients. On the other hand, cholesterol reloading partially reverses the drug effects. Methyl-beta-cyclodextrin treatment alters the colocalization of Cav2.1 with the proteins of the exocytic machinery and also impairs calcium influx in nerve terminals. These results show that lipid microdomains in presynaptic terminals are important in organizing membrane sites specialized for synaptic vesicle exocytosis. The cholesterol-enriched microdomains contribute to optimizing the compartmentalization of exocytic machinery and the calcium influx that triggers synaptic vesicle exocytosis.