The purpose of this study was to determine whether heterogeneity in endothelium-dependent responses to acetylcholine between canine blood vessels of different anatomical origin reflects variations in endothelial function or in responsiveness of vascular smooth muscle cells. Experiments were conducted in a bioassay system, where segments of femoral artery or vein with endothelium were perfused intraluminally and the perfusate used to superfuse rings of femoral arteries or veins without endothelium. Indomethacin was present in all experiments to prevent the synthesis of prostanoids. The blood vessels were contracted by phenylephrine. Measurement of wall tension in both the perfused segment and bioassay ring allowed simultaneous detection of endothelium-derived relaxing factor(s) released abluminally (segment) and intraluminally (ring). Intraluminal infusion of acetylcholine (ACh) induced relaxations in the perfused artery but not in vein segments. During arterial superfusion ACh induced relaxation in femoral arterial rings but contraction in venous rings. After treatment with atropine the arterial perfusate evoked relaxations in venous rings. Infusion of ACh through the femoral vein evoked only moderate relaxations in arterial rings. These data demonstrate that depressed endothelium-dependent relaxation to ACh in femoral veins compared to femoral arteries is due to a masking effect of the direct stimulating action of ACh and decreased release of the same mediator or the release of a different relaxing factor from venous endothelium.