Vasomotor responses to intraarterial administration of acetylcholine are mediated by release of nitric oxide, prostaglandins, and an unidentified hyperpolarizing factor from vascular endothelial cells. The contribution of endothelium-derived hyperpolarizing factor (EDHF) to the vasodilatory response to acetylcholine in the skeletal muscle circulation of patients with congestive heart failure (CHF) has not been previously characterized. Accordingly, to specifically assess the role of EDHF, the regional vascular effects of sequential administration of acetylcholine and nitroglycerin in the brachial artery were determined in the forearm circulation with strain-gauge venous occlusion plethysmography in patients with CHF and in normal subjects during combined systemic inhibition of cyclooxygenase activity with indomethacin and regional inhibition of nitric oxide synthase activity with l-N^G-monomethylarginine (l-NMMA). After administration of indomethacin, infusion of l-NMMA significantly decreased the forearm blood flow response to acetylcholine in normal subjects (5.4 ± 1.2 to 3.5 ± 0.6 ml/min/100 ml, p < 0.05) but not in patients with CHF (5.7 ± 1.3 to 5.7 ± 1.4 ml/min/100 ml). Infusion of l-NMMA did not change forearm blood flow responses to nitroglycerin in either group. The presence of a noncyclooxygenase, non-nitric-oxide relaxing factor indicates that EDHF, rather than nitric oxide, may be the predominant endothelium-derived substance mediating vasodilation in response to acetylcholine in patients with CHF.