System B(0) activity accounts for the majority of intestinal and kidney luminal neutral amino acid absorption. An amino acid transport system, called ATB(0) (also known as ASCT2), with functional characteristics similar to those of system B(0), has been recently cloned. We generated polyclonal antibodies to human and rabbit ATB(0) COOH-terminal peptides and used Western blot analysis to detect ATB(0) protein in rabbit tissues, rabbit ileal brush-border membrane vesicles (BBMV), and HeLa cells transfected with plasmids containing ATB(0) cDNAs. Immunohistochemistry was used to localize ATB(0) in rabbit kidney and intestine. In Western blots of rabbit tissues, ATB(0) was a broad smear of 78- to 85-kDa proteins. In transfected HeLa cells, ATB(0) appeared as a smear consisting of 57- to 65-kDa proteins. The highest expression was found in the kidney. ATB(0) was enriched in rabbit ileal BBMV and in HeLa cells transfected with ATB(0) cDNAs. In the kidney and in the intestine, ATB(0) was confined to the brush-border membrane (BBM) of the proximal tubular cell and of the enterocyte, respectively. Tissue and intracellular distribution of ATB(0) protein parallels that of system B(0) activity. ATB(0) protein could be the transporter responsible for system B(0) in the BBM of epithelial cells.