Objective. A number of tight junction (TJ) proteins contributing to epithelial barrier function have been identified, including occludin, tricellulin and several members of the claudin family. Whereas claudin-3 is located in the majority of epithelia including skin, lung, kidney and intestine, information on functional properties is scarce. Methods. Claudin-3 was cloned and overexpressed into the low resistance kidney tubule cell line MDCK II. Expression of claudin-3 and possible changes in other TJ proteins were checked by Western blot and immunofluorescent stainings, which were analyzed by confocal microscopy. Functional properties of claudin-3 were analyzed in detail, employing two-path impedance spectroscopy, biionic and dilution potential measurements, and flux studies with paracellular markers of different sizes. Results. Overexpression of human claudin-3 cDNA in MDCK II cells led to a marked increase in transepithelial resistance which was caused by a strongly elevated paracellular resistance. Claudin-3-transfected cells exhibited decreased permeabilities for mono- and divalent cations and for anions. The passage of macromolecular tracers up to 4,000 Da was slightly decreased, whereas the permeability to water was not affected by claudin-3 transfection. Conclusion. Claudin-3 proves to be a barrier-forming TJ protein which seals the paracellular pathway against the passage of all sorts of ions, thereby contributing to the paracellular barrier function of epithelia.