The transport proteins that mediate gamma-aminobutyric acid (GABA) reuptake have been major targets for the development of agents to treat neurological diseases such as epilepsy, where augmentation of GABAergic function is indicated. The recent isolation of cDNAs for four distinct brain GABA carriers has provided an avenue for creating more specific and selective antagonists of GABA transport. An LLC-PK1 cell line stably expressing GABA transporter type 3 (GAT-3), a beta-alanine-sensitive neuronal GABA transporter, has been generated and used to examine the kinetics, ion dependence, and pharmacological properties of the transporter. In this cell line, the GAT-3 carrier transports GABA with an apparent Km of 4 microM and a Vmax of 1.25 x 10(-16) mol/cell/min. beta-Alanine is a relatively potent inhibitor of GAT-3 GABA transport, with a K(i) value of 34 microM. beta-Alanine also serves as a substrate for the carrier (Km = 29 microM, Vmax = 1.82 x 10(-16) mol/cell/min) and appears to interact with the transporter at the same or a similar site as GABA. Other experimental GABA transport antagonists developed as anticonvulsant agents, including tiagabine, Cl-966, SKF-100330-A, SKF-89976-A, and NO-711, are weak inhibitors of GAT-3 GABA transport, suggesting that their therapeutic effects may be more related to their ability to block GABA transporters other than GAT-3. GAT-3 exhibits a sigmoidal dependence on Na+ concentration, with a Hill coefficient of 1.65, suggesting that more than one Na+ ion is involved in the transport mechanism. In contrast, the transport activity shows a hyperbolic Cl- dependence, with a Hill coefficient of 1.05. The Km for Cl- is 78 mM, a value severalfold higher than has been noted for another cloned GABA carrier, GABA transporter type 1. Interestingly, for GAT-3 a reduction of the Cl- concentration results in a small but consistent increase in the apparent Km for GABA, suggesting that the interaction of chloride with the transporter may be an important initial event in the mechanism of transport. These results underscore the unique properties of GAT-3 and distinguish this transporter as a new target for the development of GABA-mimetic agents.