Intercellular communication through gap junctions coordinates vascular tone by the conduction of vasomotor responses along the vessel wall. Gap junctions in arterioles are composed of different connexins (Cxs) (Cx40, Cx37, Cx45, Cx43), but it is unknown whether Cxs are interchangeable. We used mice with a targeted replacement of Cx40 by Cx45 (Cx40KI45) to explore whether Cx45 can functionally replace Cx40 in arterioles. Arterioles were locally stimulated using acetylcholine, bradykinin, adenosine, and K⁺ in the cremaster of Cx40KI45, Cx40-deficient (Cx40ko), and wild-type mice, and diameter changes were assessed by intravital microscopy. Additionally, arterial pressure was measured by telemetry and Cx expression verified by immunofluorescence. Acetylcholine initiated a local dilation of a similar amplitude in all genotypes (≈50%), which was rapidly conducted to upstream sites (1200 μm distance) without attenuation in wild type. In marked contrast, the remote dilation was significantly reduced in Cx40ko (25±3%) and Cx40KI45 (24±2%). Likewise, dilations initiated by bradykinin application were conducted without attenuation up to 1200 μm in wild type but not in Cx40ko and Cx40KI45. Adenosine-induced dilations and K⁺-induced constrictions were conducted similarly with decaying amplitude in all genotypes. Arterial pressure was strongly elevated in Cx40ko (161±1 versus 116±2 mm Hg) but only moderately in Cx40KI45 (133±8 mm Hg). This demonstrates that Cx40 function is critical for the conduction of acetylcholine and bradykinin dilations and cannot be substituted by Cx45. Therefore, unique properties of Cx40 are required for endothelial signal conduction, whereas nonspecific restoration of communication maintains additional functions related to blood pressure control.