Glutamine, the primary metabolic fuel for the mammalian small intestinal enterocytes, is primarily assimilated by Na-amino acid co-transporters. While Na-solute co-transport has been shown to exist in the brush border membrane (BBM) of the absorptive villus cells, the identity of Na-glutamine co-transport in rabbit small intestinal villus cells was unknown. Na-dependent glutamine uptake is present in villus BBM vesicles. An intravesicular proton gradient did not stimulate this Na-dependent glutamine uptake, while Li⁺ did not significantly suppress this uptake. These observations in concert with amino acid substitution studies suggested that Na-glutamine co-transporter in the villus cell BBM was the newly identified co-transporter B⁰AT1 (SLC6A19). Quantitative real time PCR identified the message for this co-transporter in villus cells. Thus, a full length cDNA of B⁰AT1 was cloned and expressed in MDA-MB-231 cells. This expressed co-transporter exhibited characteristics similar to that observed in villus cells from the rabbit small intestine. Antibody was generated for B⁰AT1 which demonstrated the presence of this co-transporter protein in the villus cell BBM. Kinetic studies defined the kinetic parameters of this co-transporter. Thus, this study describes the identification, cloning and characterization of the Na-amino acid co-transporter responsible for the assimilation of a critical amino acid by the absorptive villus cells in the mammalian small intestine.