Background Endothelial hyperpermeability is regulated by a myosin-II light chain (MLC2) phosphorylation-dependent contractile mechanism. Thrombin is a potent inducer of hyperpermeability of cultured monolayers of endothelial cells (ECs) via Rho kinase-mediated MLC2-phosphorylation. Aim The aim of the present study was to investigate the effects of thrombin on in situ endothelial morphology and barrier integrity. Methods Cytoskeletal dynamics, regions of paracellular flux and MLC2-phosphorylation of ECs were visualized by digital 3D imaging microscopy of pressurized rat kidney arterioles. Myosin phosphatase targeting subunit (MYPT1)-phosphorylation was used as a surrogate marker for Rho kinase activity. Results Thrombin induced the formation of F-actin filaments in ECs in situ and rounding of the ECs, in the absence of obvious formation of gaps between ECs. These changes were accompanied by an increase in MLC2-phosphorylation and a decrease in barrier integrity. In vitro analysis revealed that Rho kinase activity on F-actin filaments was associated with a contractile response that enhanced opening of the barrier. Rho kinase activity was not detectable on F-actin filaments induced by histamine, an inducer of a more transient hyperpermeability response. Inhibition of the myosin phosphatase mimicked the effects of thrombin on barrier function. The thrombin-induced changes in in situ MLC2 phosphorylation and barrier function were Rho kinase-dependent. Conclusions These data demonstrate a direct effect of thrombin on EC morphology and barrier integrity in intact microvessels. Furthermore, they establish an important contribution of enhanced Rho kinase activity to the development of prolonged but not transient types of endothelial barrier dysfunction.