Inflammatory mediators such as thrombin evoke increases in vascular permeability through activation of endothelial contractile mechanisms which involve increased levels of MLC phosphorylation catalyzed by Ca 2 + /calmodulin-dependent myosin light chain kinase (MLCK). We previously noted that the high molecular weight endothelial MLCK isoform (EC MLCK) is stably associated with a complex containing p60 s r c and 80kDa cortactin, an actin-binding protein and known p60 s r c target. In this study we have utilized in vitro binding assays to confirm specific interaction between EC MLCK and cortactin. Tyrosine phosphorylation of either EC MLCK (Y 4 6 4 , Y 4 7 1 ) or cortactin (Y 4 2 1 , Y 4 6 6 , and Y 4 8 2 ) by p60 s r c significantly increased this direct association. Site-specific antibody and peptide studies subsequently confirmed EC MLCK AA #972-979 and 1019-1025 as sites of cortactin interaction. EC MLCK-cortactin interaction in vitro failed to modulate MLCK enzymatic activity but appeared to inhibit EC MLCK binding to F-actin, while EC MLCK abolished cortactin-mediated augmentation of Arp2/3-stimulated actin polymerization. These data suggest that cortactin-EC MLCK interaction may be a novel determinant of endothelial cortical actin-based cytoskeletal rearrangement.