Mature dendritic cells (DCs) establish homo- and hetero-cellular contacts that might favour the formation of gap junctions. We studied whether connexins (Cxs), protein subunits of gap junctions, are expressed by lymph node DCs in mice under normal conditions or after skeletal muscle damage. Double immunolabelling and confocal microscopy studies revealed frequent co-localization (82 ± 9%; n = 4 experiments) of Cx45 and DEC205 in lymph node DCs, whereas Cx43 was rarely found in these cells. However, Cx43 protein was strongly up-regulated in DEC205⁺ DCs after skeletal muscle damage. Up-regulation of Cx43 gene expression by tissue damage was also demonstrated in mice carrying in one allele a β-galactosidase gene instead of the Cx43 coding region. In these mice numerous DEC205⁺ DCs expressed β-galactosidase only after skeletal muscle damage (Cx43⁺/DEC205⁺ cells were 45.0 ± 3.5 cells/10⁴ μm²; n = 2 experiments). The effect of several mixtures or individual cytokines on the expression of functional gap junctions between DCs was tested in tsDCs, a dendritic cell line (1) as well as in bone marrow-derived DCs (BMDC) in primary culture. Animals were humanely killed for the primary cell culture for bone marrow. Under control conditions, tsDCs did not communicate via gap junctions as tested with the dye coupling technique (Lucifer yellow). However, after treatment with keratinocyte-conditioned medium (48 ± 3%; n = 3 experiments) or cytokine mixtures composed of TNF-alpha/IL-1beta or TNF-alpha/IL-1beta/IFN-gamma (38 ± 5% and 43 ± 5%, respectively; n = 3 experiments for each treatment), they became transiently coupled through a pathway sensitive to octanol, a gap junction blocker. Cellular coupling induced by both pro-inflammatory cytokine mixtures was prevented by IL-6. Single cytokines (TNF-alpha, IL-1beta, IFN-gamma or IL-6) or mixtures other than the described above did not induce coupling. Increased levels of protein and mRNA of Cx43 and Cx45 accompanied the appearance of cellular coupling. Similarly, BMDCs were not dye coupled under control conditions, but after treatment with TNF-alpha/IL-1beta they became transiently coupled. This response was abrogated in cells co-treated with IL-6. These studies provide the first demonstration of Cx expression and function in DCs. Previous studies have shown that formation of homotypic clusters of DCs induced by high cell density enhance T-cell stimulation, up-regulate co-stimulatory molecules, and the transfer of antigen between clustered DCs (2). Since clustered DCs might establish gap junctional communication, we propose that these membrane channels could mediate or coordinate functions of DCs as initiator of adaptive immunity.