In cell culture a kidney epithelial cell line MDCK, forms a continuous sheet of identically oriented asymmetrical cells joined by circumferential occluding junctions. The reconstructed epithelial membrane has transport and permeability qualities of in vivo transporting epithelia. The cell layer can be readily manipulated when cultured on a freely permeable membrane filter and, when placed in an Ussing chamber, electrophysiological measurements can be taken. In the absence of a chemical gradient, the cell layer generates an electrical potential of 1.42 mV, the apical surface negative. It is an effective permeability barrier and lacks significant shunting at the clamped edge, as indicated by a resistance of 84 ohms-cm2, which increased when bulk flow from basolateral to apical was induced by an osmotic gradient or electroosmosis. The MDCK cell layer is cation selective with a relative permeability ratio, PNa/PCl, of 1.7. Net water flux, apical to basolateral, was 7.3 mul cm-2 hr-1 in the absence of a chemical gradient. The morphological and functional qualities of a transporting epithelium are stable in cell culture, and the potential use of a homogeneous cell population in cell culture would enhance studies of epithelial transport at the cellular and subcellular levels.