Neurotransmitter (NT) transporters, mainly devoted to the reuptake of NTs released at synapses, can also operate in reverse mode. Leak of NTs through this pathway has been suggested to be relevant in a number of pathological conditions, such as glutamate excitotoxicity (Rossi et al. Nature 403:316, 2000), or epileptic seizures caused by GABA spillover (Richerson & Wu, J.Neurophysiol. 90:1363, 2003). However, the conditions in which this phenomenon may take place are still controversial. In our experiments, reverse GABA transport was induced by intracellular injections of GABA in Xenopus oocytes expressing the neuronal transporter GAT1. The reverse mode of operation was assessed by measurement of outward transport currents in voltage-clamp and confirmed by [3H]GABA efflux determinations. Changes in intracellular ionic conditions affected the reverse current: higher concentrations of Na+ enhanced the ratio of outward to inward transport current;. on the contrary, a significant decrease was observed upon treatments causing a reduction of intracellular Cl-. Particularly, a significant decrease in the relative reverse transport was observed after depletion of internal Cl- caused by the activity of the coexpressed K+/Cl- symporter KCC2. These findings indicate that any condition favoring higher intracellular Cl- levels, such as underexpression of Cl- exporters, defects in osmoregulation or in ionic homeostasis, may be potentially relevant in regulating GABA leak through GAT1.