Gap junctions permit the exchange of regulatory molecules between cells and play important roles during organogenesis. The expression pattern of the gap junction proteins connexin 26, 32, and 43 was studied by immunohistochemistry in the developing, adult, and injured rat teeth. Connexins 32 and 43, but not the connexin 26, were detected during the late stages of embryonic tooth development (bell stage). Expression of connexin 32 was predominant in epithelial cells, whereas connexin 43 was more widely distributed and found in both epithelial and mesenchymal cells. During cytodifferentiation (early postnatal stages), both connexin 32 and 43 were expressed in the epithelial-derived ameloblasts, synthesizing and secreting the enamel matrix proteins. In mesenchyme, connexin 32 was observed only in differentiating odontoblasts, while connexin 43 was expressed in both differentiating and functional odontoblasts, which secrete the dentin matrix. In adult rat teeth, connexin 26 and 43 were expressed in the odontoblastic layer at low and high levels, respectively, while connexin 32 was absent from odontoblasts. Electron microscopy showed that connexin 43 was distributed exclusively at sites of contacts between odontoblasts. However, double immunostaining combined with confocal microscopy suggested an occasional overlap between odontoblasts and calcitonin gene-related peptide-positive nerve fibers. Denervation experiments showed that the expression of connexins in dental pulp was independent of innervation, whereas in injured teeth connexin 43 was upregulated in pulpal fibroblasts. Finally, cultured dental epithelial cells expressed both connexin 32 and 43, and connexin 43 was detected in cultured pulp fibroblasts in vitro, thus mimicking the in vivo distribution pattern of connexins. These results demonstrate that connexins are involved in tooth development and suggest that a given connexin may have distinct roles during odontogenesis and tooth homeostasis.