The effect of chloride ion on the transport of [3H]dopamine into synaptic vesicles purified from rat striatum has been evaluated. The inclusion of 10 mM chloride ion in the incubation medium produced a 100% increase in temperature-sensitive [3H]dopamine uptake into synaptic vesicles from approximately 1800 pmol/mg to 3600 pmol/mg of protein. Half-maximal effects were observed with chloride ion at 4 mM concentration. The anion selectivity of stimulation supports the presence of anion channels within the membranes of dopaminergic storage organelles. Low concentrations of thiocyanate ion (less than 10 mM), 4-acetamido-4'-isothiocyano-2-2'-disulfonic acid stilbene (100 microM), and duramycin (5 micrograms/ml) selectively blocked the chloride ion stimulated accumulation of [3H]dopamine. Higher concentrations of these agents are required to significantly reduce [3H]dopamine uptake in the absence of chloride ion. These results suggest that both components of the proton electrochemical gradient (delta psi and delta pH) are important for dopamine uptake by brain vesicles. This article presents the first demonstration that chloride ion plays a role in the transport of dopamine into vesicles isolated from the CNS.