1. We examined the contribution of endothelium-derived hyperpolarizing factor (EDHF) to the impairment of endothelium-dependent relaxation caused by acetylcholine (ACh) in the aorta of streptozotocin-induced diabetic rats, by using N omega-L-nitro-arginine methylester (L-NAME) and tetraethylammonium chloride (TEA) to inhibit nitric oxide (NO) and EDHF, respectively. 2. ACh-induced relaxation of the aorta decreased in diabetic rats. In contrast, sodium nitroprusside-induced relaxation was the same in diabetic rats and control rats. 3. Treatment with 5 x 10(-7) M L-NAME resulted in a right shift of the dose-response curves of ACh-induced relaxation in the aorta. The shift was greater in the control aorta. 4. Treatment with 5 x 10(-4) M TEA resulted in a similar right shift in both the control and diabetic aorta. 5. Therefore, while endothelium-derived NO appears to contribute to the impairment of ACh-induced endothelium-dependent relaxation in the aorta of diabetic rats, EDHF does not.