The small GTPase Rab13 localizes to tight junctions in epithelial cells and regulates the recruitment of claudin1 and ZO-1, two proteins required for the assembly of functional tight junctions. Rab13 directly binds to the alpha-catalytic subunit of protein kinase A (PKA alpha cat) and reversibly inhibits PKA-dependent phosphorylation of vasodilator-stimulated phosphoprotein (VASP), a key actin cytoskeletal remodeling protein. The inhibition of VASP phosphorylation abolishes the targeting of VASP to cell-cell junctions, which in turn leads to a delay in the recruitment of claudin1 and ZO1 into tight junctions. Consequently, tight junctions formed in epithelial cells expressing the GTP-bound Rab13 are structurally disorganized and functionally leaky for small molecules (A. M. Marzesco et al. [2002]. Mol. Biol. Cell13, 1819-1831; K. Kohler et al. [2004]. J. Cell Biol. 165, 175-180). Our data provide the first direct link between activation of small GTPases and the recruitment of cytoskeletal modulators into tight junctions. Here, we describe different procedures we used to demonstrate that Rab13 interacts with PKA and reversibly controls phosphorylation and recruitment of VASP.