1. Endothelium-dependent vascular regulation was investigated in mesenteric resistance arteries of Goldblatt two kidney-one clip (2K1C) renovascular hypertensive rats. 2. Third order branches of mesenteric arteries were dissected free and mounted on glass cannulae in an organ chamber. Changes in vascular diameter were measured in pressurized and perfused arteries with a video dimension analyzer. 3. Acetylcholine evoked endothelium-dependent relaxations that were much more pronounced with intraluminal than with extraluminal application. In 2K1C rats the relaxation induced by intraluminal, but not extraluminal acetylcholine was decreased compared to normotensive Wistar Kyoto rats (WKY). Increased duration of hypertension further decreased the response to intraluminal but not extraluminal acetylcholine. 4. Endothelin-1 and noradrenaline caused contractions which were augmented by removal of the endothelium. This augmentation was reduced in 2K1C rats compared to WKY; the difference was small with noradrenaline but more pronounced with endothelin-1. 5. In arteries without endothelium the sensitivity, but not the maximal contraction to endothelin-1 was lower in 2K1C rats, while the response to noradrenaline was not different in 2K1C rats and WKY. The sensitivity to the peptide was not further affected by increasing the duration of hypertension. 6. Thus, renovascular hypertension leads to an impaired intraluminal, but not extraluminal activation of the release of endothelium-derived relaxing factor and a decreased inhibitory effect of the endothelium against endothelin-1- and noradrenaline-induced contractions in mesenteric resistance arteries. Furthermore, the sensitivity, but not the maximal response of vascular smooth muscle to endothelin-1 was reduced.