Valproate is an important anticonvulsant currently in clinical use for the treatment of seizures. We used electrophysiological and tracer uptake methods to examine the effect of valproate on a ␥-aminobutyric acid (GABA) transporter (mouse GAT3) expressed in Xenopus laevis oocytes. In the absence of GABA, valproate (up to 50 mM) had no noticeable effect on the steady-state electrogenic properties of mGAT3. In the presence of GABA, however, valproate enhanced the GABA-evoked steady-state inward current in a dose-dependent manner with a halfmaximal concentration of 4.6 ؎ 0.5 mM. Maximal enhancement of the GABA-evoked current was 275 ؎ 10%. Qualitatively similar observations were obtained for human GAT1 and mouse GAT4. The valproate enhancement did not alter the Na ؉ or Cl ؊ dependence of the steady-state GABA-evoked currents. Uptake experiments under voltage clamp suggested that the valproate enhancement of the GABA-evoked current was matched by an enhancement in GABA uptake. Thus, despite the increase in GABA-evoked current, ion/GABA co-transport remained tightly coupled. Uptake experiments indicated that valproate is not transported by mouse GAT3 in the absence or presence of GABA. Valproate also enhanced the rate of the partial steps involved in transporter presteady-state charge movements. We propose that valproate increases the turnover rate of GABA transporters by an allosteric mechanism. The data suggest that at its therapeutic concentration, valproate may enhance the activity of neuronal and glial GABA transporters by up to 10%. ␥-Aminobutyric acid (GABA)1 is the most abundant inhibitory neurotransmitter in the central nervous system. Transport of GABA into cells is accomplished by Na ϩ -dependent and Cl Ϫ -facilitated GABA transporters (GATs) found in the plasma membrane of neurons and glia (1-10). Thus, the GABA transporters regulate synaptic and extra-synaptic concentrations of GABA and, in this capacity, are partly responsible for the regulation of inhibitory neurotransmission in the nervous system. Because of the inhibitory role of GABA, potentiation of GABAergic neurotransmission via inhibition or reversal of the GABA transporters is believed to have therapeutic value in treating epileptic seizures and stroke (11-13). Indeed, inhibitors of the GABA transporters are known to increase GABA levels in the brain (14 -16). These agents exhibit anticonvulsant activity in animal models, and one (tiagabine) that preferentially targets the most abundant GABA transporter isoform in the brain (GAT1) has been in clinical use since 1997 (16 -22). Several other clinically used antiepileptic drugs are reported to act, at least in part, via potentiating GABA-mediated inhibition in the brain (11, 23); however, little is known regarding the potential effect of these drugs on the GABA transporters (24).Valproate (2-propylpentanoate) has been in clinical use since 1967 and is effective against many types of epileptic seizures (both partial and generalized seizures). Although the exact mechanism of valproate action is not ...