Experiments were designed to investigate the influence of steady flow and pressure on endothelial function in the rabbit carotid artery. Increases and decreases in isometric force were compared in static rings and perfused (5 or 50 ml/min) segments of the same arteries in the presence and absence of endothelium. The alpha(1)-adrenoceptor agonist phenylephrine and the muscarinic agonist acetylcholine were applied as vasoconstrictor and vasodilator stimuli, respectively. Continuous flow (5 and 50 ml/min) reduced the cGMP content and shifted the concentration-response curve to phenylephrine to the left compared with nonperfused static rings. Removal of the endothelium abolished the differences in cGMP content and the sensitivity to phenylephrine between static rings and perfused segments. No difference in sensitivity to phenylephrine was observed in tissues treated with N(omega)-nitro-l-arginine methyl ester (l-NAME). Acetylcholine-evoked relaxations were increased in perfused segments. l-NAME nearly abolished the acetylcholine-evoked relaxation in static rings, whereas about one-half of the relaxation remained in segments exposed to flow. This remnant relaxation was blocked by inhibition of endothelial small- and intermediate-conductance calcium-activated potassium channels by apamin plus 1-[(2-chlorophenyl)diphenylmethyl]-1H-pyrazole (TRAM-34). These experiments demonstrate that continuous flow increases the constriction evoked by alpha(1)-adrenergic activation in the rabbit carotid artery through a reduced influence of basally released endothelial NO and, furthermore, that luminal flow unmasks an ability of the endothelium to release a non-NO, noncyclooxygenase vasodilator, presumably endothelium-derived hyperpolarizing factor.