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Objective: Synaptic vesicles (SVs) are essential organelles that store and release neurotransmitters within presynaptic terminals. Isolated and purified SVs can be used to investigate their biochemical, structural, and functional characteristics, to elucidate the neurotransmission process. This paper describes a method for obtaining purified and functional SVs. Methods: SVs were isolated from the brains of Sprague--Dawley rats using differential and density-gradient centrifugation. Their morphology was observed with electron microscopy. The amount of protein in the SV fraction was determined by the Bradford assay. Specific SVs were purified using magnetic beads and immunoisolation, and immunoblot assays were used to measure levels of SV markers. SV glutamate uptake was assayed by radiotracer efflux assays. Results: A total of 10 mg SV protein was purified from 22 g of hippocampal and cerebral cortex tissue. The purified SVs were homogenous small spherical vesicles with a diameter of ~40 nm as assessed with electron microscopy, and they were rich in the protein SV protein 2 (SV2), vesicular glutamate transporter 1 (VGLUT1), and VGLUT2, but not VGLUT3. The SV2-specific SVs obtained by immunoadsorption with nearly 83% yield could uptake L- glutamate. Rose Bengal, a VGLUT inhibitor, prevented SV2-specific SVs from taking-up L- glutamate. Conclusions: This study describes a successful method for obtaining purified SVs that retained their structure and ability to perform their primary in vivo functions. These SVs can be used to understand SV composition and function, and the process of neurotransmitter release, and to evaluate therapeutic drugs for nervous system diseases. [ABSTRACT FROM AUTHOR]