Objective: Tight junctions are multicomponent structures, with claudin proteins defining paracellular permeability. Claudin 3 is a candidate for the exceptional “tightness” of human urothelium, being localised to the terminal tight junction (TJ) of superficial cells. Our aim was to determine whether claudin 3 plays an instigating and/or a functional role in the urothelial TJ.Materials and Methods: Normal human urothelial (NHU) cells maintained as non-immortalised cell lines were retrovirally-transduced to over-express or silence claudin 3 expression. Stable sublines induced to stratify or differentiate were assessed for TJ formation by immunocytochemistry and transepithelial electrical resistance (TER). Expression of claudin 3, ZO-1 and ZO-1α+ was examined in native urothelium by immunohistochemistry.Results: Claudin 3 expression was associated with differentiation and development of a tight barrier and along with ZO-1 and ZO-1α+ was localised to the apical tight junction in native urothelium. Knockdown of claudin 3 inhibited formation of a tight barrier in three independent cell lines, however, overexpression of claudin 3 was not sufficient to induce tight barrier development in the absence of differentiation. A differentiation-dependent induction of the ZO-1α+ isoform was found to coincide with barrier formation. Whereas claudin 3 overexpression did not induce the switch to co-expression of ZO-1α-/ZO-1α+, claudin 3 knockdown decreased localisation of ZO-1 to the TJ and resulted in compromised barrier function.Conclusions: Urothelial cytodifferentiation is accompanied by induction of claudin 3 which is essential for the development of a terminal TJ. A coordinated switch to the ZO-1α+