Endothelium-dependent relaxations are depressed in hypertension. In this study we investigated the possible involvement of endothelium-dependent smooth muscle hyperpolarization in this phenomenon. In isolated aortic segments from control rats, acetylcholine (10−8-10−5M) elicits relaxations after precontraction with norepinephrine (10−7M), and acetylcholine or carbachol (10−5M) induce smooth muscle hyperpolarization (10.6±0.9 mV). Both effects disappear after removal of the endothelium and are depressed by tetraethylammonium (3±10−3M), a rather nonspecific blocker of K+channels, but not by glibenclamide (10−5M), a potent blocker of the ATP-regulated K+channels, which has a marked effect on the relaxation induced by BRL 38227. The relaxation effect of acetylcholine is impaired in norepinephrine-contracted preparations from hypertensive rats but is not further depressed by tetraethylammonium. In aorta from hypertensive rats, hyperpolarization induced by carbachol was significantly reduced to a mean of only 21.8% of the values obtained in preparations from normotensive rats. From the relaxation-hyperpolarization relation obtained with BRL 38227 (opening K+channels), it is derived that the endothelium-dependent hyperpolarization (∼10 mV) contributes for at least 20–30% of the maximal relaxation effect of acetylcholine on rat aorta. It is concluded that the diminished endothelium-dependent hyperpolarization may contribute to the depression of the endothelium-dependent relaxation in hypertension.