The vesicular inhibitory amino acid transporter (VIAAT) is a synaptic vesicle protein responsible for the vesicular storage of gamma-aminobutyrate (GABA) and glycine which plays an essential role in GABAergic and glycinergic neurotransmission. The transport mechanism of VIAAT remains largely unknown. Here, we show that proteoliposomes containing purified VIAAT actively took up GABA upon formation of membrane potential (Deltapsi) (positive inside) but not DeltapH. VIAAT-mediated GABA uptake had an absolute requirement for Cl(-) and actually accompanied Cl(-) movement. Kinetic analysis indicated that one GABA molecule and two Cl(-) equivalents were transported during one transport cycle. VIAAT in which Glu(213) was specifically mutated to alanine completely lost the ability to take up both GABA and Cl(-). Essentially the same results were obtained with glycine, another substrate of VIAAT. These results demonstrated that VIAAT is a vesicular Cl(-) transporter that co-transports Cl(-) with GABA or glycine in a Deltapsi dependent manner. It is concluded that Cl(-) plays an essential role in vesicular storage of GABA and glycine.