An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance developed within 4-6 h after plating and achieved a steady-state value of 104 +/- 1.8 omega-cm2 after 24 h. Mature monolayers were morphologically and functionally polarized. They contained junctional complexes composed of desmosomes and tight junctions with properties similar to those of "leaky" epithelia. Monolayers were capable of maintaining a spontaneous electrical potential sensitive to amiloride, produced a net water flux from the apical to basal side, and discriminated between Na+ and Cl- ions. The MDCK permeability barrier behaves as a "thin" membrane with negatively charged sites. It has: (a) a linear conductance/concentration relationship; (b) an asymmetric instantaneous current/voltage relationship; (c) a reduced ability to discriminate between Na+ and Cl- caused by lowering the pH; and (d) a characteristic pattern of ionic selectivity which suggests that the negatively charged sites are highly hydrates and of medium field strength. Measurements of Na+ permeability of electrical and tracer methods ruled out exchange diffusion as a mechanism for ion permeation and the lack of current saturation in the I/deltapsi curves does not support the involvement of carriers. The discrimination between Na+ and Cl- was severely but reversibly decreased at low pH, suggesting that Na+-specific channels which exclude Cl- contain acidic groups dissociated at neutral pH. Bound Ca++ ions are involved in maintaining the integrity of the junctions in MDCK monolayers as was shown by a reversible drop of resistance and opening of the junctions in Ca++-free medium containing EGTA. Several other epithelial cell lines are capable of developing a significant resistance under the conditions used to obtain MDCK monolayers.