It is assumed that the Na+-independent transport of sulfate (S) in the mammalian gastrointestinal tract (GIT) is like in the renal proximal tubule, e.g., mediated by CFEX/slc26a6 and sat-1/slc26a1 across the apical and serosal membrane, respectively. However, the exact characteristics of S transport and localization of these proteins in the GIT epithelium are not known. In this work we performed uptake studies in sat-1-expressing oocytes, to define the sat-1 functional properties, and PCR, Western blotting (WB) and immunocytochemical (IC) studies in the rat GIT, to reveal its expression in the epithelium. In oocytes, the transporter acted as an exchanger for S, bicarbonate, and oxalate with apparent affinities of 0.36±0.16, 3.5±2.0, and 0.049±0.026 mM, respectively. Glycolate and glyoxylate trans-stimulated S uptake from 81±12 to 201±14 and 117±7 pmol/h?oocyte. By PCR, sat-1 mRNA was present in all parts of the GIT. In WB of isolated GIT cell membranes the anti-sat-1 antibody labeled a major ~80 kDa protein band, whereas IC studies revealed a granular pattern of intracellular staining in oxyntic cells of the stomach and in enterocytes of the small and large intestine. Endosomal localization of sat-1 was disproven. In rat GIT, sat-1 localization in peroxisomes is discussed.