ABSTRACT Platelet endothelial cell adhesion molecule‐1 (PECAM‐1), an immunoglobulin family vascular adhesion molecule, is involved in endothelial cell migration and angiogenesis (1, 2). We found that endothelial cells lacking PECAM‐1 exhibit increased single cell motility and extension formation but poor wound healing migration, reminiscent of cells in which Rho activity has been suppressed by overexpressing a GTPase‐activating protein (3). The ability of PECAM‐1 to restore wound healing migration to PECAM‐1‐deficient cells was independent of its extracellular domain or signaling via its immunoreceptor tyrosine‐based inhibitory motif. PECAM‐1‐deficient endothelial cells had a selective defect in RhoGTP loading, and inhibition of Rho activity mimicked the PECAM‐1‐deficient phenotype of increased chemokinetic single cell motility at the expense of coordinated wound healing migration. The wound healing advantage of PECAM‐1‐positive endothelial cells was not only Rho mediated but pertussis toxin inhibitable, characteristic of migration mediated by heterotrimeric G‐protein‐linked seven‐transmembrane receptor signaling such as signaling in response to the serum sphingolipid sphingosine‐1‐phosphate (S1P) (4, 5). Indeed, we found that the wound healing defect of PECAM‐1 null endothelial cells is minimized in sphingolipid‐depleted media; moreover, PECAM‐1 null endothelial cells fail to increase their migration in response to S1P. We have also found that PECAM‐1 localizes to rafts and that in its absence heterotrimeric G‐protein components are differentially recruited to rafts, providing a potential mechanism for PECAM‐1‐mediated coordination of S1P signaling. PECAM‐1 may thus support the effective S1P/RhoGTP signaling required for wound healing endothelial migration by allowing for the spatially directed, coordinated activation of Galpha signaling pathways.—Gratzinger, D., Canosa, S., Engelhardt, B., Maori, J. A. Platelet endothelial cell adhesion molecule‐1 modulates endothelial cell motility through the small G‐protein Rho, FASEB J. 17, 1458–1469 (2003)