A disruption of endothelial barrier function is a predisposing factor for the development of atherosclerosis. The phosphorylation of myosin light chain (MLC) is a key signal of barrier dysfunction. MLC phosphorylation is thought to take place sequentially at S19 and then T18, induces actin stress fiber formation and generates traction force to disrupt cell-cell contact. However, it is unclear how the phosphorylation at two sites contributes to barrier dysfunction. The present study investigated the role of mono- and di-phosphorylation of MLC (MLC-P and MLC-PP) in thrombin-induced barrier dysfunction. Thrombin (1 u/mL) increased the cytosolic Ca2+ concentration and decreased the transendothelial electrical resistance (TEER) with a peak at 1 and 3-5 min, respectively, in porcine aortic endothelial cells (PAEC). A new SDS-PAGE method using a Phos-tagTM compound, which specifically binds to a phosphate group, allowed separate quantification of MLC-P and MLC-PP. PAEC at confluence contained 25% MLC-P and 2% MLC-PP under resting conditions. Thrombin stimulation only slightly increased MLC-P at 3 min, while it showed up on stress fibers. MLC-PP increased to a peak of 35% at 3-5 min, and thereafter declined to the resting level within 15 min. MLC-PP was localized in the subjunctional region of cell-cell contact in addition to the stress fibers at 3 min. ROCK inhibitors, Y27632 and H1152, inhibited the thrombin-induced decrease in TEER, the increase in MLC-PP and the subjunctional localization of MLC-PP, while having no effect on the Ca2+ response. The MLC-P level remained unaffected by Y27632, while the stress fiber localization was inhibited. Gq inhibitor, YM254890, inhibited the Ca2+ response and the decrease in TEER, but it had no effect on the increase in MLC-PP. The introduction of dominant negative mutants of G12, G13 and Gq using a cell-penetrating peptide inhibited the thrombin-induced decrease in TEER. The present study provides the first evidence that MLC-P and MLC-PP are differentially regulated in endothelial cells, and that Ca2+-independent, ROCK-mediated MLC-PP in the subjunctional regions plays a critical role in the thrombin-induced barrier dysfunction. Inhibition of MLC-PP is thus necessary to restore endothelial barrier dysfunction.