Endothelial cells (EC) actively participate in lymphocyte transendothelial migration by remodeling their actin cytoskeleton. We studied the endothelial cell abluminal matrix receptor (focal adhesion, FA) complexes to determine if these structures were remodeled following lymphocyte adhesion. Lymphocytes (PBL) were isolated from whole blood and added to cultured EC. Lectin‐stimulated PBL adhered to EC spontaneously, whereas adhesion of freshly isolated lymphocytes to EC was induced by pre‐treatment with MCP‐1 or activating anti‐CD11a mAb. Sustained adhesion between lymphocytes and EC resulted in a significant, contact‐dependent decrease in paxillin incorporation into the FA following 15, but not 5, min of contact. EC FA remodeling was associated with increased phosphorylation of pp125 FA kinase. Pretreatment of the EC with an activating β1 integrin monoclonal antibody, TS2/16, prevented lymphocyte‐stimulated FA remodeling. Further, TS2/16 pretreatment inhibited transendothelial migration of lymphocytes and β1 integrin‐deficient JY lymphoblasts. These data demonstrate that sustained lymphocyte adhesion induces remodeling of EC FA structures and that this remodeling event is required for efficient lymphocyte transendothelial migration in vitro.