Rxt1/NTT4 (SLC6A17) belongs to a gene family of "orphan transporters" whose substrates and consequently functions remain unidentified. Although Rxt1/NTT4 was previously thought to function as a sodium-dependent plasma membrane transporter, recent studies localized the protein to synaptic vesicles of glutamatergic and GABAergic neurons. Here, we provide evidence indicating that Rxt1/NTT4 functions as a vesicular transporter selective for proline, glycine, leucine, and alanine. Using Western blot, immunoprecipitation, immunocytochemistry, and polymerase chain reaction approaches, we demonstrate that PC12 cells express the Rxt1/NTT4 gene and protein. Small interfering RNA (siRNA)-mediated knockdown of Rxt1/NTT4 in PC12 cells resulted in selective reductions in uptake levels for proline, glycine, leucine, and alanine. Likewise, gas chromatography analysis of amino acid content in an enriched synaptic vesicle fraction from wild-type and siRNA-Rxt1/NTT4 PC12 cells revealed that proline, glycine, leucine, and alanine levels were decreased in siRNA-treated cells compared with wild-type cells. Furthermore, Rxt1/NTT4-transfected Chinese hamster ovary (CHO) cells exhibited significant uptake increases of these amino acids compared with mock-transfected CHO cells. Finally, proline uptake in both PC12 cells and Rxt1/NTT4-transfected CHO cells was dependent on the electrochemical gradient maintained by the vacuolar-type H(+)-ATPase. These data indicate that the orphan Rxt1/NTT4 protein functions as a vesicular transporter for proline, glycine, leucine, and alanine, further suggesting its important role in synaptic transmission.