NO is an important modulator of endothelial barrier function. The mechanism by which it reduces agonist-induced permeability is not well understood. Here it was tested whether NO attenuates hyperpermeability by inactivation of the contractile machinery, an important determinant of barrier function, which is regulated by a balance of myosin light chain kinase (MLCK) and MLC phosphatase activity (MLCP). In cultured HUVEC thrombin (Thr: 0.2U/ml) increased permeability (albumin flux) by 290%, isometric force (IT: cells cultured on collagen gels) by 34%, MLC phosphorylation (MLC~P) by 180%, MLCK activity by 63%, RhoA (pull down) by 400%, and phosphorylation of the MLCP targeting subunit (MYPT1~P, Western blot) by 180% (n=5;P<0.05), indicating that Thr activates contractile machinery via activation of MLCK and inhibition of MLCP. UO126 (10?M), an inhibitor of MEK, reduced Thr-induced MLC~P and MLCK activity, but it does not affect RhoA and MYPT1~P, indicating that MEK/ERK is an upstream activator of MLCK but not of MLCP. The NO donor spermineNONOate (100mM) attenuated Thr-induced hyperpermeability, IT, MLC~P, activation of RhoA, MEK/ERK, and MYPT1~P. In addition NO stimulated translocation of the phosphatase catalytic subunit PP1 to the MYPT1-myosin complex. These data show that NO attenuates Thr-induced barrier failure by an inactivation of the MLCK and activation of the MLCP.