Conduction of changes in diameter plays an important role in the coordination of peripheral vascular resistance, and thereby, in the control of arterial blood pressure. It is thought that conduction of vasomotor signals relies on the electrotonic spread of changes in membrane potential from a site of stimulation through gap junctions connecting the cells of the vessel wall. To explore this idea, we stimulated a short segment of mouse cremasteric arterioles with the application via micropipette of acetylcholine (ACh), an endothelium-dependent vasodilator, or pinacidil, an ATP-sensitive K⁺ channel opener. The vasodilations were evaluated at the stimulation site (local) and at 500, 1000 and 2000 µm upstream. The vasodilator response evoked by direct arteriolar hyperpolarization induced by pinacidil decayed rapidly with distance, as expected for the passive spread of an electrical signal. Deletion of the gap junction proteins connexin 37 or connexin 40 did not alter the conduction of pinacidil-induced vasodilation. In contrast to pinacidil, the vasodilator response activated by ACh spreads along the entire vessel without decrement. Although the ACh-induced conducted vasodilation was similar in wild type and connexin 37 knockout mice, deletion of connexin 40 converted the non-decremental conducted response activated by ACh into one similar to that of pinacidil, with a decline in magnitude along the vessel length. These results suggest that ACh activates a mechanism of regenerative conduction of vasodilator responses. Connexin 40 is essential for the ACh-activated regenerative vasodilator mechanism. However, this connexin or connexin 37 are not indispensable for the electrotonic spread of hyperpolarizing signals.