The low-affinity (Km = 100-200 μM) γ-aminobutyric acid (GABA) transporter from membrane vesicles from rat brain has been characterized and found to be in many aspects similar to the well-known sodium- and chloride-coupled high-affinity γ-aminobutyric acid transporter (Km = 2-4 μM). Influx by this system is sodium and chloride dependent and stimulated by an interior negative membrane potential. Steady-state levels obtained by both systems are lowered by the sodium channel openers veratridine and aconitine. However, while the channel blocker tetrodotoxin fully reverses this inhibition with the high-affinity system, this is not the case for its low-affinity counterpart. Furthermore, the toxin from the scorpion Androctonus australis Hector inhibited high-affinity transport only. Efflux of γ-aminobutyric acid taken up by the high-affinity system displayed a Km of about 100 μM. Exchange catalyzed by the low-affinity system was observed in the absence of external sodium and chloride. Furthermore, both activities copurified in the fractionation procedure developed to purify the high-affinity transporter. All these observations are consistent with the idea that both activities are manifestations of only one γ-aminobutyric acid transporter. The high-affinity binding site represents the extracellular and the low-affinity site the cytosolic aspect of the transporter. In addition, it was found that right-side-out synaptosomes also contain a low-affinity GABA transporter. This apparently represents a different transport protein.