—Connexins have been hypothesized to play an important role in intercellular communication within the vascular wall and may provide a mechanistic explanation for conduction of vasomotor responses. To test this hypothesis, we studied the transmission of vasomotor responses in the intact skeletal muscle microcirculation of connexin40-deficient mice (Cx40^−/−). Arterioles were locally stimulated with hyperpolarizing dilators (acetylcholine [ACh] as well as bradykinin [Bk]) or depolarizing K⁺ solution, and the resulting changes in diameter were measured using a videomicroscopy technique at the site of application and up to 1.32 mm upstream. Arterial pressure was elevated 25% in Cx40^−/− mice (94±5 versus 75±4 mm Hg). Vessels selected for study had equivalent basal diameter and vasomotor tone in both genotypes of mice. Vasomotion was present in small arterioles of both genotypes, but its intensity was exaggerated in Cx40^−/− mice. ACh and Bk induced dilation (33% and 53%, respectively, of maximal response) at the site of application that was of similar magnitude in both genotypes. These dilations were observed to spread upstream within <1 second without significant attenuation in Cx40^+/+ mice. However, spreading was severely attenuated in Cx40^−/− animals (11±4% versus 35±7% with ACh and 38±5% versus 60±7% with Bk in Cx40^−/− and Cx40^+/+, respectively; P<0.05). In contrast, conducted vasoconstrictions, induced by K⁺ solution decreased equally with distance in both genotypes. These results support a significant role for Cx40 in vascular intercellular communication. Our observations indicate that Cx40 is required for normal transmission of endothelium-dependent vasodilator responses and may underlie altered vasomotion patterns.