The vascular endothelium constitutes a highly effective fluid and solute barrier through the regulated apposition of tight junction protein complexes between adjacent endothelial cells. As endothelial cell-mediated functions and pathology are sensitive to hemodynamic forces (cyclic strain and shear stress), we hypothesised a dynamic regulatory link between endothelial tight junction assembly/function and hemodynamic stimuli. We have investigated this hypothesis via examination of the precise effects of cyclic strain on the expression, modification, and function o f occludin and ZO-1, pivotal components o f the tight junction complex. Moreover, the mechanotransduction pathway by which tight junction regulation occurs was also investigated. For these studies, cultured bovine aortic endothelial cells (BAECs) were subjected to physiological levels o f equibiaxial cyclic strain (0 or 5% strain, 60 cycles/min, 24 h). In response to strain, protein expression o f both occludin and ZO-1 increased. Increased mRNA levels were also observed for occludin, but not ZO -1. These changes were accompanied by tyrosine dephosphorylation of occludin and serine/threonine phosphorylation of ZO-1, modifications that could be completely blocked by pharmacological inhibition with dephostatin (tyrosine phosphatase) and rottlerin (protein kinase C), respectively. The effects o f cyclic strain on the association and subcellular localisation o f occludin and ZO-1 was also investigated. In response to cyclic strain we observed a significant increase in endothelial occludin/ZO-1 co-association in parallel with increased localisation of both proteins to the cell-cell border. Moreover, these events were completely blocked by dephostatin and rottlerin and were accompanied by a strain-dependent reduction in transendothelial permeability to FITC-dextran, an event that could also be blocked by inhibitor addition indicating a causal relationship between biochemical changes and barrier function. We observed that these modifications o f the tight junction following strain were mediated via Gi-proteins, the small GTPase Rac-1, and the signaling molecule p38. Overall, these findings indicate that physiological cyclic strain up-regulates vascular endothelial tight junction assembly, with subsequent consequences for barrier integrity, putatively via tyrosine phosphatase- and PKC-dependent modification of occludin and ZO-1. The signal is transduced via G-proteins, Rac-1 and p38.