We have cloned the Na(+)-dependent neutral amino acid transporter B0 (ATB0) from rabbit jejunum and from the human intestinal cell line Caco-2. Rabbit intestinal ATB0 (riATB0) cDNA codes for a protein of 541 amino acids with 10 potential transmembrane domains. When expressed in HeLa cells, riATB0 mediates the transport of several neutral amino acids, including glutamine, in a Na(+)-dependent manner. Anionic amino acids, cationic amino acids, and N-methylated amino acids are excluded by riATB0. When expressed in Xenopus laevis oocytes, riATB0 increases the transport of neutral amino acids severalfold. The induced transport activity is specific for neutral amino acids, with no noticeable interaction with anionic, cationic, and N-methylated amino acids. However, riATB0 does interact with anionic amino acids at acidic pH. In oocytes expressing riATB0, the neutral amino acid threonine evokes inward currents at a holding potential of -50 mV. The amino acid-evoked current is sensitive to membrane potential. The inward current increases as the membrane potential is hyperpolarized, but the current reverses at about -30 to -40 mV. Threonine evokes outward currents if the membrane potential is depolarized beyond this value. We have also cloned the ATB0 from the human intestinal cell line Caco-2. The Caco-2 ATB0 cDNA also codes for a protein of 541 amino acids that is essentially identical to the ATB0 expressed in the human choriocarcinoma cell line JAR. Reverse transcription-polymerase chain reaction (RT-PCR) and restriction analysis of the RT-PCR products indicate that the human intestine and the human kidney proximal tubular cell line HKPT express an ATB0 identical to the ATB0 expressed in Caco-2 cells.