Synaptic vesicles (SVs) in the central nervous system upon stimulation undergo rapid calcium-triggered exo-endocytic cycling within the nerve terminal which at least in part depends on components of the clathrin- and dynamin-dependent endocytosis machinery. How exocytic SV fusion and endocytic retrieval are temporally and spatially coordinated is still an open question. One possibility is that specialized membrane microdomains characterized by their high content in membrane cholesterol may assist in the spatial coordination of synaptic membrane protein recycling. Quantitative proteomic analysis of detergent-resistant lipid microdomains (DRMs) isolated from rat brain synapses or cholesterol-depleted control samples by liquid chromatography tandem mass spectrometry identified a total of 159 proteins. Among these 122 proteins were classified as cholesterol-dependent DRM or DRM-associated proteins, many of which with proven or hypothesized functions in exo-endocytic vesicle cycling including clathrin, the clathrin adaptor complex AP-2, and a variety of SV proteins. In agreement with this SV membrane and endocytic proteins display a partial resistance to extraction with cold Triton X-100 in cultured rat hippocampal neurons where they co-localize with labeled cholera toxin B, a marker for cholesterol-enriched DRMs. Moreover, SV proteins form cholesterol-dependent complexes in CHAPS-extracted synaptic membrane lysates. Our combined data suggest that lipid microdomains may act as spatial coordinators for exo-endocytic vesicle cycling at synapses.