Pure synaptic vesicles have been isolated in sucrose-KCl media. They are able to take up calcium in the presence of ATP and Mg. This is based on the following evidence. First, the synaptic vesicle fraction is the gradient peak for calcium uptake. Second, it was not possible to separate ACh and ATP from the uptake peak after refractionation of synaptic vesicles. Third, the fraction appears very pure on morphological and biochemical grounds. The physiological significance of the calcium uptake by synaptic vesicles is discussed.