Vascular endothelial cells are coupled by gap junctions that permit cell-to-cell transfer of small molecules, including signals that may be important for vasomotor responses. Connexin37 (Cx37) and connexin40 (Cx40) are the predominant gap-junction proteins present in mouse endothelium. We examined the effect of eliminating Cx37, Cx40, or both, on interendothelial communication in mouse aorta. Intercellular transfer of biocytin and[2-(4-nitro-2,1,3-benzoxadiazol-7-yl)aminoethyl]trimethylammonium (NBD-TMA)was used to assess gap-junction-mediated coupling. Ablation of Cx40 generally had a greater effect on dye-transfer than ablation of Cx37. The effect of Cx40 ablation on dye-transfer was age dependent. There was a 27-fold reduction in biocytin transfer in embryonic Cx40^–/– aortic endothelium, a much larger change than in aortas of 6-7-week-old Cx40^–/– animals, which showed a 3.5-fold reduction. By contrast, there was no reduction in biocytin transfer in embryonic Cx37^–/– endothelium. Embryonic aortas lacking both Cx37 and Cx40 showed a complete loss of endothelial dye-transfer. Surprisingly,elimination of Cx40 resulted in up to a 17-fold drop in endothelial Cx37 on western blots, whereas deletion of Cx37 reduced endothelial Cx40 up to 4.2-fold. By contrast, in the medial layer, both Cx37 and Cx43 increased∼fourfold in Cx40^–/– aortas. Declines in non-ablated endothelial connexins were not mediated by changes in connexin mRNA levels, suggesting a post-transcriptional effect. Our results indicate that Cx37 and Cx40 are the only functional connexins expressed in mouse aortic endothelium and are collectively crucial for endothelial communication. Furthermore, Cx37 and Cx40 are codependent on each other for optimal expression in vascular endothelium.