Endothelial cell junctional adhesion molecule (JAM)-C has been proposed to regulate neutrophil migration. In the current study, we used function-blocking mAbs against human JAM-C to determine its role in human leukocyte adhesion and transendothelial cell migration under flow conditions. JAM-C surface expression in HUVEC was uniformly low, and treatment with inflammatory cytokines TNF-α, DL-1β, or LPS did not increase its surface expression as assessed by FACS analysis. By immunofluorescence microscopy, JAM-C staining showed sparse localization to cell-cell junctions on resting or cytokine-activated HUVEC. Surprisingly, staining of detergent-permeabilized HUVEC revealed a large intracellular pool of JAM-C that showed little colocalization with von Willebrand factor. Adhesion studies in an in vitro flow model showed that functional blocking JAM-C mAb alone had no inhibitory effect on polymorphonuclear leukocyte (PMN) adhesion or transmigration, whereas mAb to ICAM-1 significantly reduced transmigration. Interestingly, JAM-C-blocking mAbs synergized with a combination of PECAM-1, ICAM-1, and CD99-blocking mAbs to inhibit PMN transmigration. Overexpression of JAM-C by infection with a lentivirus JAM-C GFP fusion protein did not increase adhesion or extent of transmigration of PMN or evoke a role for JAM-C in transendothelial migration. These data suggest that JAM-C has a minimal role, if any, in PMN transmigration in this model and that ICAM-1 is the preferred endothelial-expressed ligand for PMN β2 integrins during transendothelial migration. Copyright © 2007 by The American Association of Immunologists, Inc.