The characteristics of the hyperpolarization response to acetylcholine (ACh) in endothelial cells from the guinea pig coronary artery were studied by microelectrode recording technique. ACh (30 nM to 3 μM) induced membrane hyperpolarization in a dose-dependent manner. The sustenance of the response required the presence of external calcium. The hyperpolarization was not affected by nifedipine (1 μM) but was inhibited by the potassium channel blockers charybdotoxin (10 nM), tetraethylammonium (1 mM), and 4-aminopyridine (0.5 mM). Glibenclamide (10 μM) and apamin (1 μM) were not effective. The inhibitors of endothelium-derived relaxing factor/nitric oxide synthesis Nω-nitro L-arginine (50 μM) andNG-monomethyl l-arginine (30 μM) had no effect on the resting membrane potential or the ACh-induced responses. No hyperpolarization was observed with application of sodium nitroprusside (10 μM) or 8-bromo-cGMP (0.1 μM). Ouabain (10 μM) depolarized the membrane significantly by 5 mV, but the ACh hyperpolarization was not affected. Indomethacin (10 μM) was without effect on the resting membrane potential or the hyperpolarization to ACh. These results show that ACh-induced hyperpolarization is dependent on external calcium and can be inhibited by certain potassium channel blockers. The hyperpolarization response is not mediated by endothelium-derived relaxing factor/nitric oxide, cGMP, a cyclooxygenase product, or stimulation of the Na-K pump.