Recent studies have shown that gap junctions (GJ) can mediate the transfer of molecules between leukocytes and endothelial cells in culture. However, the role of GJ in the inflammatory response remains controversial. Some authors show that GJ blockade facilitates transmigration but others have reported that GJ communication is not important in this process. We studied the role of GJ on TNF-α induced leukocyte adhesion to venular endothelium in vivo. Since there are no specific blockers to GJ, we conducted these experiments on endothelial cell specific connexin43-deficient mice (Cx43eKO) and connexin40-deficient mice (Cx40KO). We evaluated the time course of leukocyte adhesion and transmigration through the venular endothelium of the mouse cremaster microcirculation observed by intravital microscopy, under basal conditions or 60 min after an intrascrotal application of TNF-α(0.5 mg in 0.3 ml saline). Animals were anesthetized with pentobarbital sodium (80 mg kg^-1 I.P.), maintained under deep anaesthesia with supplementary doses of this drug during the observation period, and sacrificed with an anaesthetic overdose (500 mg kg^-1 I.P.) at the end of the experiment. Surprisingly, in basal conditions Cx43eKO mice (n=3) showed greater leukocyte adhesion and transmigration than wild type mice (n=5; 11.3 ± 2.6 vs 4.8 ± 0.5 cells per 100 μm vessel length, and 13.3 ± 3.5 vs 5.7 ± 0.7 cells per 10⁴ μm² perivenular area, respectively, means ± SEM). In wild type mice, over a period of four hours following application of TNF-α, the number of adherent leukocytes increased approximately three times (from 7.8 ± 1.9 to 26.6 ± 3.5 cells per 100 μm) and the number of transmigrated leukocytes increased up to four times above control levels (from 8.6 ± 1.0 to 26.2 ± 3.0 cells per 10⁴ μm²). In contrast, TNF-α stimulation in Cx43eKO mice caused a moderate increase in the number of adhered leukocytes (from 13.5 ± 2.0 to 20.3 ± 4.4 cells per 100 μm), remaining below TNF-α-stimulated wild type animals. Furthermore, TNF-α did not modify leukocyte transmigration in Cx43eKO mice (16.1 ± 2.6 vs 16.1 ± 2.0 cells per 10⁴ μm²). Reduced adhesion and transmigration were observed in Cx40KO mice (n=2) compared to wild type animals both, in basal conditions and in response to TNF-α stimulation. These results support our hypothesis that GJ participate in the processes of leukocyte activation, adhesion and transmigration during inflammation.