Barrier epithelia such as the renal collecting duct (in the absence of antidiuretic hormone) and thick ascending limb, as well as the stomach and mammalian bladder, exhibit extremely low permeabilities to water and small nonelectrolytes. A cell culture model of such epithelia is needed to determine how the structure of barrier apical membranes reduce permeability and how such membranes may be generated and maintained. In the present studies, the transepithelial electrical resistance and isotopic water and urea fluxes were measured for Madin-Darby canine kidney (MDCK) type I and type II cells, as well as type I cells expressing the mucin protein, MUC1, in their apical membranes. Although earlier studies had found the unstirred layer effects too great to permit measurement of transepithelial permeabilities, use of ultrathin semipermeable supports in this study overcame this difficulty. Apical membrane diffusive water permeabilities were 1.8 +/- 0.4 x 10(-4) cm/s and 3.5 +/- 0.5 x 10(-4) cm/s in MDCK type I and type II cells, respectively, at 20 degrees C. Urea permeability in type I cells at the same temperature was 6.0 +/- 0.9 x 10(-6) cm/s. These values resemble those of other barrier epithelial apical membranes, either isolated or in intact epithelia, and the water permeability values are far below those of other epithelial cells in culture. Transfection of MDCK type I cells with the major human urinary epithelial mucin, MUC1, led to abundant expression of the fully glycosylated form of the protein on immunoblots, and flow cytometry revealed that virtually all the cells expressed the protein. However, MUC1 had no effect on water or urea permeabilities. In conclusion, MDCK cells grown on semipermeable supports form a model barrier epithelium. Abundant expression of mucins does not alter the permeability properties of these cells.