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Synaptic vesicles isolated from guinea-pig cerebral cortex had an electrophoretic mobility of −3.55μm·s-1·V-1·cm in saline–sorbitol, pH7.2, at 25°C (ionic strength 0.015g-ions/1). The mobility was pH-dependent, varied with ionic strength and indicated that the vesicular surface contained weak acidic functions with a pKa in the range 3.0–3.8. Although the vesicular surface was determined to be highly negatively charged, treatment with neuraminidase had no effect on mobility and indicated that the relatively strong carboxyl groups of sialic acid do not contribute significantly to vesicular electrokinetic properties. Treatment of synaptic vesicles with trypsin or trypsinized concanavalin A resulted in increases in mobility, but treatment with ribonuclease, deoxyribonuclease, chrondroitinase ABC or hyaluronidase had no significant effect on mobility. Mn2+or Ca2+was more effective in decreasing vesicle mobility than was Mg2+, Sr2+or Ba2+. The electrokinetic properties of the synaptic vesicle surface are discussed and contrasted with the properties of the synaptosomal membrane.