Rab13 is recruited to tight junctions from a cytosolic pool after cell-cell contact formation. Tight junctions are intercellular junctions that separate apical from basolateral domains and are required for the establishment/maintenance of polarized transport in epithelial cells. They form selective barriers regulating the diffusion of ions and solutes between cells. They also maintain the cell surface asymmetry by forming a "fence" that prevents apical/basolateral diffusion of membrane proteins and lipids in the outer leaflet of the plasma membrane. We generate stable MDCK cell lines expressing inactive (T22N mutant) and constitutively active (Q67L mutant) Rab13 as GFP-Rab13 chimeras. Expression of GFP-Rab13Q67L delays the formation of electrically tight epithelial monolayers, induces the leakage of small nonionic tracers from the apical domain, and disrupts the tight junction fence diffusion barrier. It also alters the tight junction strand structure and delays the localization of the tight junction transmembrane protein, claudin1. In contrast, the inactive Rab13T22N mutant does not disrupt tight junction functions, tight junction strand architecture, or claudin1 localization. Here we describe a set of assays that allows us to investigate the role of Rab13 in modulating tight junction structure and function.