PECAM-1 (CD31) is a cellular adhesion and signaling receptor that is highly expressed at endothelial cell-cell junctions in confluent vascular beds. Previous studies have implicated PECAM-1 in the maintenance of vascular barrier integrity; however, the mechanisms behind PECAM-1-mediated barrier protection are still poorly understood. The goal of the present study, therefore, was to examine the pertinent biological properties of PECAM-1 (i.e. adhesion and/or signaling) that allow it to support barrier integrity. We found that, compared with PECAM-1-deficient endothelial cells, PECAM-1-expressing endothelial cell monolayers exhibit increased steady-state barrier function, as well as more rapid restoration of barrier integrity following thrombin-induced perturbation of the endothelial cell monolayer. The majority of PECAM-1-mediated barrier protection was found to be due to the ability of PECAM-1 to interact homophilically and become localized to cell-cell junctions, because a homophilic binding-crippled mutant form of PECAM-1 was unable to support efficient barrier function when re-expressed in cells. By contrast, cells expressing PECAM-1 variants lacking residues known to be involved in PECAM-1-mediated signal transduction exhibited normal to near-normal barrier integrity. Taken together, these studies suggest that PECAM-1-PECAM-1 homophilic interactions are more important than its signaling function for maintaining the integrity of endothelial cell junctions.