The conduction of vasodilation along resistance vessels has been presumed to reflect the electrotonic spread of hyperpolarization from cell to cell along the vessel wall through gap junction channels. However, the vasomotor response to acetylcholine (ACh) encompasses greater distances than can be explained by passive decay. To investigate the underlying mechanism for this behavior, we tested the hypothesis that ACh augments the conduction of hyperpolarization. Feed arteries (n = 23; diameter, 58 ± 4 µm; segment length, 2-8 mm) were isolated from the hamster retractor muscle, cannulated at each end, and pressurized to 75 mmHg (at 37°C). Vessels were impaled with one or two dye-containing microelectrodes simultaneously (separation distance, 50 µm to 3.5 mm). Membrane potential (E_m) (rest, approximately 30 mV) and electrical responses were similar between endothelium and smooth muscle, as predicted for robust myoendothelial coupling. Current injection (0.8 nA, 1.5 s) evoked hyperpolarization (10 ± 1 mV; membrane time constant, 240 ms) that conducted along the vessel with a length constant () = 1.2 ± 0.1 mm; spontaneous E_m oscillations (~1 Hz) decayed with  = 1.2 + 0.1 mm. In contrast, ACh microiontophoresis (500 nA, 500 ms, 1 µm tip) evoked hyperpolarization (14 ± 2 mV) that conducted with  = 1.9 ± 0.1 mm, 60% further (P < 0.05) than responses evoked by purely electrical stimuli. These findings indicate that ACh augments the conduction of hyperpolarization along the vessel wall.