Na,K-ATPase regulates a variety of transport functions in epithelial cells. In cultures of human retinal pigment epithelial (RPE) cells, inhibition of Na,K-ATPase by ouabain and K(+) depletion decreased transepithelial electrical resistance (TER) and increased permeability of tight junctions to mannitol and inulin. Electrophysiological studies demonstrated that the decrease in TER was due to an increase in paracellular shunt conductance. At the light microscopy level, this increased permeability was not accompanied by changes in the localization of the tight junction proteins ZO-1, occludin, and claudin-3. At the ultrastructural level, increased tight junction permeability correlated with a decrease in tight junction membrane contact points. Decreased tight junction membrane contact points and increased tight junction permeability were reversible in K(+)-repletion experiments. Confocal microscopy revealed that in control cells, Na,K-ATPase was localized at both apical and basolateral plasma membranes. K(+) depletion resulted in a large reduction of apical Na,K-ATPase, and after K(+) repletion the apical Na,K-ATPase recovered to control levels. These results suggest a functional link exists between Na,K-ATPase and tight junction function in human RPE cells.