To assess the hypothesis that gap junctions (GJs) participate on leukocyte-endothelium interactions in the inflammatory response, we compared leukocyte adhesion and transmigration elicited by cytokine stimulation in the presence or absence of GJ blockers in the hamster cheek pouch, and also in the cremaster muscle of wild type (WT) and endothelium-specific Cx43 null mice (Cx43e^/-). In the cheek pouch, topical tumor necrosis factor-alpha (TNF-, 150 ng/ml, 15-min) caused a sustained increment in the number of leukocytes adhered to venular endothelium (LAV) and located at perivenular regions (LPV). Superfusion with the GJ blockers 18--glycyrrhetinic acid (AGA, 75µM) or 18--glycyrrhetinic acid (50µM) abolished the TNF--induced increase in LAV and LPV; carbenoxolone (75µM), or oleamide (100µM), reduced LAV by 50% and 75%, respectively, and LPV to a lesser extent. None of these GJ blockers modified venular diameter, blood flow, or leukocyte rolling. In contrast, glycyrrhizin (75µM) a non-GJ blocker analogue of AGA was devoid of effect. Interestingly, when AGA was removed 90-min after TNF- stimulation, LAV started to rise at a similar rate as in control. Conversely, application of AGA 90-min post TNF- reduced the number of previously adhered cells. In WT mice, intrascrotal injection of TNF- (0.5µg/0.3ml) increased LAV (four-fold) and LPV (three-fold) as compared to saline-injected controls. In contrast to the observations in wild type animals, TNF- stimulation did not increase LAV or LPV in Cx43e^/- mice. These results demonstrate an important role for gap junctional communication in leukocyte adhesion and transmigration during acute inflammation in vivo, and further suggest that endothelial Cx43 is key in these processes.