Muscarinic receptor agonists have primarily been used to characterize endothelium-dependent vasodilator dysfunction with overweight/obesity. Reliance on a single class of agonist however, yields limited, and potentially misleading, information regarding endothelial vasodilator capacity. The aims of this study were to determine: 1) if the overweight/obesity-related reduction in endothelium-dependent vasodilation extends beyond muscarinic receptor agonists; and 2) the contribution of nitric oxide to endothelium-dependent vasodilation is reduced in overweight/obese adults. Eighty-six middle-aged and older adults were studied: 42 normal weight (age 54±1 yr; 21 M/21 F; BMI 23.4±0.3 kg/m²) and 44 overweight/obese (54±1 yr; 28 M/16 F; 30.3±0.6 kg/m²). Forearm blood flow (FBF) responses to intra-arterial infusions of acetylcholine in the absence and presence of the endothelial nitric oxide synthase inhibitor NG-monomethyl-L-arginine (L-NMMA), methacholine, bradykinin, substance P, isoproterenol and sodium nitroprusside were measured by strain-gauge plethysmography. FBF responses to each endothelial agonist were significantly blunted in the overweight/obese adults. Total FBF (area under the curve) to acetylcholine (50±5 vs 79±4 mL/100 mL tissue), methacholine (55±4 vs 86±5), bradykinin (62±5 vs 85±4 mL/100 mL tissue), substance P (37±4 vs 57±5 mL/100 mL tissue) and isoproterenol (62±4 vs 82±6 mL/100 mL tissue) were 30%-40% lower in the overweight/obese vs normal weight adults. L-NMMA significantly reduced the FBF response to acetylcholine to the same extent in both groups. There were no differences between the groups in the FBF responses to SNP. These results indicate that agonist-stimulated endothelium-dependent vasodilation is universally impaired with overweight/obesity. Moreover, this impairment appears to be independent of nitric oxide.