The high-affinity mammalian brain L-proline transporter (PROT) belongs to the GAT1 gene family, which includes Na- and Cl-dependent plasma membrane carriers for neurotransmitters, osmolites, and metabolites. These transporters couple substrate flux to transmembrane electrochemical gradients, particularly the Na gradient. In the nervous system, transporters clear synapses and help to replenish transmitters in nerve terminals. The localization of PROT to specific excitatory terminals in rat forebrain suggests a role for this carrier in excitatory transmission (Renick et al., 1999). We investigated the voltage regulation and electrogenicity of this novel transporter, using human embryonic kidney (HEK) 293 cells stably transfected with rat PROT cDNA. In physiological solutions between -140 and - 40 mV, L-proline (PRO) and its six-member ring congener L-pipecolate (PIP) induced inward current. The current-voltage relationship and the variance of current fluctuations were similar for PRO- and PIP-induced current, and the ratio of induced variance to the mean current ranged from 20 to 60 fA. Des- Tyr-Leu-enkephalin (GGFL), a competitive peptide inhibitor of PROT, reduced the rat PROT-associated current to control levels. GGFL alone did not elicit currents, and the GGFL-sensitive substrate-induced current was absent in nontransfected cells. Finally, GGFL inhibited PROT-mediated transport only when applied to the extracellular face of PROT. These data suggest that (1) PROT uptake is electrogenic, (2) individual transporter currents are voltage- independent, and (3) GGFL is a nonsubstrate inhibitor that interacts either with an extracellular domain of PROT or in an externally accessible pore.