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Synaptic vesicles (SV) are secretory organelles that store neurotransmitters in presynaptic nerve endings.In this report, differential centrifugation separation SVs from homogenates of human and animal brain tissues and purification by density gradient ultracentrifugation are described.Isolation protocols for SVs can be divided into two groups.The first group involves the preparation of isolated nerve terminals (synaptosomes) by differential centrifugation.The synaptosomes are subsequently lysed to release the SVs.The second group of protocols involves direct isolation of SV from brain homogenates.Purification methods include: (1) Gradient fractionation using Nycodenz: SV preparation was recovered from homogenate suspended in 35% (w/v) Nycodenz and overlaid by 30%, 25%,20%, 15%, 10% Nycodenz.The gradients were centrifuged at 120,000 g for 17 h.(2)Glycerol velocity sedimentation: Layer a pad of 0.5 ml of sucrose, on the top of the pad, layer a 5% to 25% continuous glycerol gradient.Load the SV preparation on the top of the gradient,Ultracentrifuge 75 min at 220,000 g.(3) Self-generated iodixanol gradient: The SV preparation was adjusted to 14% (w/v) iodixanol and centrifuged at 265,000 g in a vertical rotor for 4 h.(4) Discontinuous iodixanol sedimentation gradient: The SV preparation was layered over 5%, 10%, 15% and 20% iodixanol.After centrifugation at 140,000 g for 16 h the SVs banded at the 5% / 10% iodixanol interface.(5) Gradient fractionation using 100%, 85%,70% D2O, Ultracentrifuge 1.5 hr at 225,000 g.These procedures allow for the rapid isolation of highly pure SVs from brain homogenates.