The vasoactive protease thrombin is a known activator of the protease-activated receptor-1 (PAR1) via cleavage of its N-terminus. PAR1 activation stimulates the RhoA / Rho kinase signaling cascade, leading to myosin light chain phosphorylation, actin stress fiber formation and changes in endothelial monolayer integrity. Previous studies suggest that some elements of this signaling pathway are localized to caveolin-containing cholesterol-rich membrane domains. Here we show that PAR1 and key components of the PAR-associated signaling cascade localize to membrane rafts and caveolae in bovine aortic endothelial cells (BAEC). To investigate the functional significance of this localization, BAEC were pretreated with filipin (5µg/mL, 5 min) to ablate lipid rafts prior to thrombin (100nM) or PAR agonist stimulation. We found that di-phosphorylation of myosin light chain (MLC) and the actin stress fiber formation normally induced by PAR activation were attenuated after lipid raft disruption. To target caveolae specifically, we used an siRNA approach to knock-down caveolin-1 expression. Thrombin-induced MLC phosphorylation and stress fiber formation was not altered in caveolin-1 depleted cells, suggesting that lipid rafts, but not necessarily caveolae, modulate thrombin-activated signaling pathways leading to alteration of the actin cytoskeleton in endothelial cells.