After exocytosis, synaptic vesicles rapidly endocytose and recycle but little is known about the molecular mechanisms involved. Rab5 is a ubiquitous low molecular weight GTP-binding protein required for endosomal fusion in fibroblasts. We have now raised polyclonal and monoclonal antibodies to rat Rab5 and show that in rat brain, Rab5 is a major synaptic vesicle protein. Immunoisolation of vesicular organelles from brain with antibodies to either Rab3A and Rab5 as small GTP-binding proteins or with synaptophysin as general synaptic vesicle marker demonstrates that there are overlapping populations of synaptic vesicles containing either Rab5 or Rab3A or both, suggesting a stage-specific association of these low-molecular weight GTP-binding proteins with synaptic vesicles. Our data provide the first biochemical evidence that synaptic vesicle recycling involves an endosomal intermediate similar to that of the receptor-mediated endocytosis pathway.