Acetylcholine (ACh)-mediated endothelium-dependent vasodilation has been shown to be impaired in obese adults. However, the mechanisms responsible for this impairment are not clear. We determined whether the blunted forearm vasodilator response to acetylcholine with obesity is due, at least in part, to reduced muscarinic receptor responsiveness. Twenty-eight sedentary middle-aged adults were studied: 14 normal weight (BMI, 23.6 ± 0.5 kg/m2) and 14 obese (32.2 ± 0.9 kg/m2). Forearm blood flow (FBF) was determined in response to intraarterial infusion of acetylcholine (8-128 μg/min) and sodium nitroprusside (SNP: 2.0-8.0 μg/min). Regardless of the dose, forearm blood flow responses to acetylcholine were 25% (P < 0.01) lower in the obese (from 4.2 ± 0.3 to 12.0 ± 0.8 mL/100 mL tissue/min) compared with normal weight (4.4 ± 0.3 to 16.9 ± 1.0 mL/100 mL tissue/min) adults. Of note, forearm blood flow responses to acetylcholine plateaued at doses higher than 32 μg/min in both groups, no further increase in forearm blood flow was observed at either 64 or 128 μg/min. EC50 for acetylcholine-stimulated vasodilation was not different between the obese (7.8 ± 0.8 μg/min) and normal weight (7.8 ± 0.6 μg/min) adults. There were no group differences in the vasodilator response to sodium nitroprusside. These results indicate that the obesity-related impairment in acetylcholine-mediated vasodilation in the human forearm is not due to reduced muscarinic receptor responsiveness or sensitivity.