The new member of the mammalian amino acid/auxin permease family, PAT2, has been cloned recently and represents an electrogenic proton/amino acid symporter. PAT2 and its paralog, PAT1/LYAAT-1, are transporters for small amino acids such as glycine, alanine, and proline. Our immunodetection studies revealed that the PAT2 protein is expressed in spinal cord and brain. It is found in neuronal cell bodies in the anterior horn in spinal cord and in brain stem, cerebellum, hippocampus, hypothalamus, rhinencephalon, cerebral cortex, and olfactory bulb in the brain. PAT2 is expressed in neurons positive for the N-methyl-d-aspartate subtype glutamate receptor subunit NR1. PAT2 is not found in lysosomes, unlike its paralog PAT1, but is present in the endoplasmic reticulum and recycling endosomes in neurons. PAT2 has a high external proton affinity causing half-maximal transport activation already at a pH of 8.3, suggesting that its activity is most likely not altered by physiological pH changes. Transport of amino acids by PAT2 activity is dependent on membrane potential and can occur bidirectionally; membrane depolarization causes net glycine outward currents. Our data suggest that PAT2 contributes to neuronal transport and sequestration of amino acids such as glycine, alanine, and/or proline, whereby the transport direction is dependent on the sum of the driving forces such as substrate concentration, pH gradient, and membrane potential.