Because the pulmonary alveolar epithelium separates air spaces from a fluid-filled compartment, it is expected that this barrier would be highly resistant to the flow of solutes and water. Investigation of alveolar epithelial resistance has been limited due to the complex anatomy of adult mammalian lung. Previous efforts to study isolated alveolar epithelium cultured on porous substrata yielded leaky monolayers. In this study, alveolar epithelial cells isolated from rat lungs and grown on tissue culture-treated Nucleopore filters resulted in tight monolayers with transepithelial resistance greater than 2,000 omega.cm2. Changes in bioelectric properties of these alveolar epithelial monolayers in response to ouabain, amiloride, and terbutaline are consistent with active sodium transport across a polarized barrier. 22Na flux measurements under short-circuit conditions directly confirm net transepithelial absorption of sodium by alveolar epithelial cells in the apical to basolateral direction, comparable to the observed short-circuit current (4.37 microA/cm2). The transport properties of these tight monolayers may be representative of the characteristics of the mammalian alveolar epithelial barrier in vivo.