Previous experiments have shown that an amino acid transport system B⁰⁺ transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B⁰⁺ transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB⁰⁺. Northern blot analysis revealed that mCATB⁰⁺ was selectively expressed in the large intestine. In situ hybridization showed the mCATB⁰⁺ mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB⁰⁺ exhibited a Na⁺-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B⁰⁺. In vivo [³H]glycine uptake assay demonstrated that a system B⁰⁺-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB⁰⁺ may absorb amino acids from the intestinal contents in the colon.