1 In the smooth muscle cells of canine coronary artery, acetylcholine (ACh) produced a transient, endothelium-dependent hyperpolarization of the membrane. A similar hyperpolarization was also elicited by exposure to Krebs solution after incubation of the artery in K-free solution for 30 min. 2 A hyperpolarization of reproducible amplitude was generated when ACh was applied at intervals greater than 30 min. Repetitive application of ACh at 15 min intervals caused a successive reduction in the amplitude of hyperpolarization. 3 The reduction in the amplitude of relaxation during five successive applications of ACh at 15 min intervals was less than 10% of the first relaxation. 4 The ACh-induced hyperpolarization was blocked by atropine but not by ouabain, whereas the K-free induced hyperpolarization was blocked by ouabain. In low Na (Li-substituted) solution, ACh still induced a hyperpolarization but the K-free induced hyperpolarization was absent. 5 In coronary artery precontracted by high-K solution, ACh produced an endothelium-dependent relaxation, without membrane hyperpolarization. The associated relaxation was resistant to ouabain but sensitive to atropine. 6 It is concluded that in the canine coronary artery, the electrogenic Na-K pump does not contribute to the endothelium-dependent hyperpolarization or relaxation. The results are consistent with the release of two different inhibitory factors from the vascular endothelium.