N-Ethylmaleimide-sensitive factor (NSF) mediates docking and/or fusion of transport vesicles in the multi-pathways of vesicular transport. NSF was highly expressed in brain and adrenal gland. Immunostaining of cerebellum with an anti-NSF monoclonal antibody showed that NSF is predominantly localized in the molecular layers and the glomeruli of the granule cell layers. This distribution coincided well with that of synaptophysin, a marker protein of synaptic vesicles. Purification and immunoprecipitation revealed that NSF is associated with brain synaptic vesicles. The present results suggest that NSF is associated with synaptic vesicles without Ca2+ influx.