1. The effects of ascorbate were assessed on vasodilatation mediated by endothelium-derived hyperpolarizing factor (EDHF) in the ciliary vascular bed of the bovine isolated perfused eye and in the rat isolated perfused mesenteric arterial bed. 2. In the bovine eye, EDHF-mediated vasodilator responses induced by acetylcholine or bradykinin were powerfully blocked when ascorbate (50 microM) was included in the perfusion medium for at least 120 min; with acetylcholine a normally-masked muscarinic vasoconstrictor response was also uncovered. 3. The blockade of EDHF-mediated vasodilatation by ascorbate was time-dependent (maximum blockade at 120 min) and concentration-dependent (10 - 150 microM). 4. Ascorbate (50 microM) also blocked acetylcholine-induced, EDHF-mediated vasodilator responses in the rat mesenteric arterial bed in a time-dependent manner (maximum blockade at 180 min). 5. The ability of ascorbate to block EDHF-mediated vasodilatation is likely to result from its reducing properties, since this action was mimicked in the bovine eye by two other reducing agents, namely, N-acetyl-L-cysteine (1 mM) and dithiothreitol (100 microM), but not by the redox-inactive analogue, dehydroascorbate (50 microM). 6. In conclusion, concentrations of ascorbate present in normal plasma block EDHF-mediated vasodilator responses in the bovine eye and rat mesentery. The mechanism and physiological consequences of this blockade remain to be determined.