Acetylcholine (ACh) evokes the conduction of vasodilation along resistance microvessels. However, it is not known which cell layer (endothelium or smooth muscle) serves as the conduction pathway. In isolated, cannulated feed arteries (≈70 &mgr;m in diameter at 75 mm Hg; length ≈4 mm) of the hamster retractor muscle, we tested the hypothesis that endothelial cells provide the pathway for conduction. Microiontophoresis of ACh (500 ms, 500 nA) onto the distal end of a feed artery evoked hyperpolarization (−13±2 mV) of both cell layers with vasodilation (15±1 &mgr;m) along the entire vessel. To selectively damage endothelial cells (confirmed by loss of vasodilation to ACh and labeling of disrupted cells with propidium iodide), an air bubble was perfused through a portion of the vessel lumen, or a 70-kDa fluorescein-conjugated dextran (FCD) was illuminated within a segment (300 &mgr;m) of the lumen. After endothelial cell damage, hyperpolarization and vasodilation conducted up to, but not through, the treated segment. To selectively damage smooth muscle cells (confirmed by loss of vasoconstriction to phenylephrine and labeling with propidium iodide), FCD was perifused around the vessel and illuminated. Vasodilation and hyperpolarization conducted past the disrupted smooth muscle cells without attenuation. We conclude that endothelial cells provide the pathway for conducting hyperpolarization and vasodilation along feed arteries in response to ACh.