CaT1 is a highly selective calcium entry channel that has been proposed to be responsible for apical calcium entry in the vitamin D-regulated transcellular pathway of Ca(2+) absorption; however, the lack of a CaT1 antibody suitable for immunohistochemistry has prevented the direct testing of this hypothesis by the localization of CaT1 protein in the gastrointestinal tract and other tissues. In this study, we developed two CaT1 antibodies and have used them to establish for the first time that CaT1 localizes to the apical membrane of intestinal absorptive cells, thereby providing the first direct evidence that this protein is in fact an apical entry channel in the gastrointestinal tract. In addition, we found that CaT1 protein is highly expressed in a number of exocrine organs including pancreas, prostate, and mammary gland, suggesting an, as yet, unrecognized role in secretory epithelia. Finally, we found CaT1 protein to be present at elevated levels in comparison with normal tissues in a series of prostate, breast, thyroid, colon, and ovarian carcinomas, consistent with previous reports of up-regulation of CaT1 mRNA in prostate cancer tissues. Our findings indicate that CaT1 is likely to serve as a component of transcellular calcium transport mechanisms in many tissues and epithelial cancers.