1. Experiments were designed to determine whether endothelium-dependent relaxing factor (s) released by acetylcholine from the canine femoral artery influences the membrane potential of coronary arterial smooth muscle. 2. The membrane potential was recorded in small canine coronary arteries (internal diameter less than or equal to 500 micron; without endothelium) by means of intracellular microelectrodes. The organ bath also contained a strip of left descending coronary artery without endothelium in which isometric force was measured to bioassay relaxing factor (s) as well as segments of femoral artery with endothelium, which served as the source of endothelium-derived relaxing factor (s). 3. Acetylcholine induced endothelium-dependent, transient hyperpolarizations and relaxations that were not affected by indomethacin. 4. Inhibition of the sodium-potassium pump by ouabain or potassium-free solution did not inhibit the relaxation to acetylcholine but prevented the corresponding hyperpolarization. 5. Activation of the sodium-potassium pump of the smooth muscle cells by readmission of potassium ions after incubation in potassium-free solution caused relaxation and marked hyperpolarization. 6. These results suggest that endothelium-derived relaxing factor (s) induces hyperpolarization of vascular smooth muscle of the canine coronary artery, possibly by activation of sodium-potassium pumping, but that this effect on the cell membrane may only partially explain endothelium-dependent relaxations evoked by acetylcholine.