The role of tight junctions (TJs) in regulating epithelial cell proliferation in conjuction with scaffolding zonula occludens (ZO) proteins has come to light recently. We examined the influence of each ZO protein (ZO-1, -2 and -3) on kidney collection duct (CD) cell proliferation. All tree ZO proteins are expressed in native CD and are present at both intercellular junctions and nuclei of cultures CD principal cells MCCDc11). Suppression of either ZO-1 or ZO-2 resulted in increased G0/G1 retention. ZO-2 suppression decreased cyclin D1 abundance whilw ZO-1 suppression increased nuclear p21 localization, the depletion of which restores cell density and relied on the presence of ZO-1. ZO-3 depletion increased cell detachment with increased cyclin D1 abundance, altered ß1 –integrin subcellular distribution and decrased occludin expression. These data reveal diverging, but interconnected, roles for each ZO protein in mCCDc11 proliferation.