Since sphingosine 1-phosphate (Sph-l-P) is stored in abundant amounts in blood platelets and released extracellularly upon stimulation, it is important to clarify the effects of this bioactive lysophospholipid on vascular endothelial cells from the viewpoint of platelet-endothelial cell interactions. In this study, we investigated the effects of Sph-l-P on the cytoskeletal remodeling of human umbilical vein endothelial cells (HUVECs). Of a focal adhesion kinase (FAK) family of non-receptor protein-tyrosine Mnases, HUVECs were found to express FAK, but scarcely proline-rich tyrosine kinase 2. Sph-l-P induced FAK tyrosine phosphorylation, myosin light chain phosphorylation, and the formation of stress fibers in HUVECs. The specific Rho rnactivator C3 transferase from Clostridium botulinum abolished all of these cytoskeletal responses induced by Sph-l-P, while pertussis toxin only partly inhibited FAK tyrosine phosphorylation, and hardly affected myosin light chain phosphorylation and stress fiber formation. In contrast, Sph-l-P-induced intracellular Ca2+ mobilization was suppressed by pertussis toxin, but not at all by C3 exoenzyme. Our results suggest that Sph-l-P, a bioactive lipid released from activated platelets, induces endothelial cell cytoskeletal reorganization, mainly through Rho-mediated signaling pathways