Background & Aims: Amino acid (and related drug) absorption across the human small intestinal wall is an essential intestinal function. Despite the revelation of a number of mammalian genomes, the molecular identity of the classic Na+-dependent imino acid transporter (identified functionally in the 1960s) remains elusive. The aims of this study were to determine whether the recently isolated complementary DNA hPAT1 (human proton-coupled amino acid transporter 1), or solute carrier SLC36A1, represents the imino acid carrier; the Na+-dependent imino acid transport function measured at the brush-border membrane of intact intestinal epithelia results from a close functional relationship between human proton-coupled amino acid transporter-1. and Na+/H+ exchanger 3 (NHE3). Methods: PAT1 function was measured in isolation (Xenopus laevis oocytes) and in intact epithelia (Caco-2 cell monolayers and rat small intestine) by measurement of amino acid and/or H+ influx. Tissue and membrane expression of PAT1 were determined by reverse-transcription polymerase chain reaction and immunohistochemistry. Results: PAT1-specific immunofluorescence was localized exclusively to the luminal membrane of Caco-2 cells and human and rat small intestine. The substrate specificity of hPAT1 is identical to that of the imino acid carrier. In intact epithelia, PAT1-mediated amino acid influx is reduced under conditions in which NHE3 is inactive. Conclusions: The identification in intact epithelia of a cooperative functional relationship between PAT1 (H+/amino acid symport) and NHE3 (Na+/H+ exchange) explains the apparent Na+ dependence of the imino acid carrier in studies with mammalian intestine. hPAT1 is the high-capacity imino acid carrier localized at the small intestinal luminal membrane that transports nutrients (imino/amino acids) and orally active neuromodulatory agents (used to treat affective disorders).