An increase in tissue blood flow requires relaxation of smooth muscle cells along entire branches of vascular resistance networks, which are comprised of feed arteries and the arterioles they supply. Our goal is to define signalling pathways intrinsic to the endothelium which coordinate vasodilatation along feed arteries and arterioles and thereby increase tissue perfusion. In skeletal muscle, contractile activity produces vasodilatation that ‘ascends’ from arterioles into their feed arteries and is essential to attaining high levels of muscle blood flow, which is otherwise restricted by proximal resistance. Vessels of the heart and brain respond similarly to an increase in metabolic demand. To study the mechanism(s) underlying this vascular communication, feed arteries supplying the retractor muscle of hamsters were isolated and pressurized (75 mmHg) to develop spontaneous myogenic tone (resting diameter, 50 to 60 µm; maximal diameter, 90 to 100 µm). Using acetylcholine (ACh) as a stimulus, we tested the hypothesis that the signal for conducted vasodilatation travels along the endothelium. A brief (≤ 1 s) pulse of ACh delivered from a micropipette onto the distal end of a feed artery (resting membrane potential, -30 to -35 mV) evoked hyperpolarization (10 to 15 mV) and vasodilatation (15 to 20 µm) that conducted rapidly (several mm/s) along the entire vessel (length, 3 to 4 mm). Following selective disruption of endothelial cells (light-dye treatment) within a segment (~250 µm long) midway along the vessel, hyperpolarization and vasodilatation spread up to but not through the damaged region. In contrast, selective smooth muscle cell damage had no effect on conducted responses. Thus, the endothelium provides a cellular pathway for conducting hyperpolarization and vasodilatation. To test the hypothesis that endothelial cells promote smooth muscle relaxation through direct electrical coupling, 2 microelectrodes were used to simultaneously impale an endothelial cell and a smooth muscle cell separated by 500 µm along the vessel. Both cells displayed equivalent membrane potentials at rest and during responses to ACh. Microinjection of negative current into either cell caused hyperpolarization of the other cell along with conducted vasodilatation, confirming that respective cell layers are electrically coupled to each other. Moreover, focal electrical field stimulation evoked similar responses, indicating that endothelial cells can be exited electrically to produce hyperpolarization and vasodilatation. Thus, electrical signals initiated by and conducted along the endothelium can be transmitted directly to the surrounding smooth muscle to evoke electromechanical relaxation. Immunolabeling for gap junction proteins revealed robust expression of connexins 37, 40 and 43 along borders of endothelial cells. Electron microscopy confirmed the presence of myoendothelial gap junctions for heterocellular transmission of electrical signals. As complementary signals (e.g. nitric oxide) have been implicated in the conduction of vasodilatation, we tested the hypothesis that Ca²⁺ waves propagate from cell to cell along the endothelium. During Ca²⁺ imaging, ACh triggered an increase in endothelial cell Ca²⁺ at the site of stimulation which preceded two distinct events: (1) a rapid synchronous decrease in smooth muscle Ca²⁺ along the entire vessel, and (2) an ensuing Ca²⁺ wave that propagated bidirectionally for > 1 mm along the endothelium at ~111 µm/s. To investigate the functional role of Ca²⁺ waves, a vessel was perifused with charybdotoxin + apamin at the site of ACh stimulation. Remarkably, this local inhibition of KCa channels (to prevent hyperpolarization) unmasked a ‘slow’ conducted vasodilatation that traveled at ~21 µm/s. Recorded 500 µm upstream from the ACh stimulus, a rise in endothelial cell Ca²⁺ preceded dilatation by ~10 s. When nitric oxide synthase and cyclooxygenase were inhibited, the slow vasomotor response was abolished while Ca²⁺ waves remained intact. Experiments performed in mice demonstrate similar behavior for arteriolar networks in vivo. Our findings collectively resolve 2 distinct yet complementary signalling pathways for the conduction of vasodilatation along microvascular endothelium in response to ACh: (1) a rapid electromechanical relaxation of smooth muscle cells along the vessel initiated by KCa channels, and (2) an ensuing slow ‘wave’ of Ca²⁺ along the endothelium that releases autacoids to promote pharmacomechanical relaxation and sustain the coordinated vasomotor response.