opathogenic Escherichia coli (EPEC) is a food-borne pathogen that causes infantile diarrhea worldwide. EPEC decreases the activity and surface expression of the key intestinal Cl Ϫ /HCO 3 Ϫ exchanger SLC26A3 [downregulated in adenoma (DRA)], contributing to the pathophysiology of early diarrhea. Little is known about the mechanisms governing membrane recycling of DRA. In the current study, Caco-2 cells were used to investigate DRA trafficking under basal conditions and in response to EPEC. Apical Cl Ϫ /HCO 3 Ϫ exchange activity was measured as DIDS-sensitive 125 I Ϫ uptake. Cell surface biotinylation was performed to assess DRA endocytosis and exocytosis. Inhibition of clathrin-mediated endocytosis by chlorpromazine (60 M) increased apical Cl Ϫ /HCO 3 Ϫ exchange activity. Dynasore, a dynamin inhibitor, also increased function and surface levels of DRA via decreased endocytosis. Perturbation of microtubules by nocodazole revealed that intact microtubules are essential for basal exocytic (but not endocytic) DRA recycling. Mice treated with colchicine showed a decrease in DRA surface levels as visualized by confocal microscopy. In response to EPEC infection, DRA surface expression was reduced partly via an increase in DRA endocytosis and a decrease in exocytosis. These effects were dependent on the EPEC virulence genes espG1 and espG2. Intriguingly, the EPEC-induced decrease in DRA function was unaltered in the presence of dynasore, suggesting a clathrin-independent internalization of surface DRA. In conclusion, these studies establish the role of clathrin-mediated endocytosis and microtubules in the basal surface expression of DRA and demonstrate that the EPEC-mediated decrease in DRA function and apical expression in Caco-2 cells involves decreased exocytosis. DRA recycling; EPEC infection; endocytosis; exocytosis; clathrin; microtubules DOWNREGULATED IN ADENOMA (DRA), or SLC26A3, is an integral membrane transporter expressed on the apical membrane of epithelial cells, including the small intestine and colon. Mutations in the SLC26A3 gene are associated with congenital chloride diarrhea (CLD), characterized by voluminous diarrhea with high fecal chloride content (28). DRA knockout mice exhibit severe diarrhea and a phenotype similar to that of CLD patients (32). Emerging studies indicate that posttranslational modifications of DRA protein may underlie the pathophysiology of diarrheal diseases. For example, CLD-causing mutations induce diarrhea by causing loss of functional DRA protein at the plasma membrane, possibly via its misfolding and mistrafficking (25,36). Studies from our laboratory have shown that enteropathogenic Escherichia coli (EPEC) infection decreases DRA function and surface levels (7), which may underlie early diarrhea; however, the cellular recycling mechanisms of DRA under normal or diseased conditions are not well understood. Thus, investigation of the cellular pathways responsible for maintaining appropriate DRA levels on the apical membrane is important for understanding disease pathogenesis...