Hg2ϩ is commonly used as an inhibitor of many aquaporins during measurements of transcellular water transport. To investigate whether it could also act on the paracellular water transport pathway, we asked whether addition of Hg 2ϩ affected transport of radiolabeled probes through tight junctions of a salivary epithelial cell monolayer. Inclusion of 1 mM Hg 2ϩ decreased transepithelial electrical resistance by 8-fold and augmented mannitol and raffinose flux by 13-fold, which translated into an estimated 44% increase in pore radius at the tight junction. These Hg 2ϩ -induced effects could be partially blocked by the protein kinase A (PKA) inhibitor N-[2-((p-bromocinnamyl) amino) ethyl]-5-isoquinolinesulfonamide, 2HCl (H89), suggesting that both-PKA dependent and PKA-independent mechanisms contribute to tight junction regulation. Western blot analyses showed a 2-fold decrease in tight junction-associated occludin after Hg 2ϩ treatment and the presence of a novel hyperphosphorylated form of occludin in the cytoplasmic fraction. These findings were corroborated by confocal imaging. The results from this study reveal a novel contribution of the PKA pathway in Hg 2ϩ