This study sought to better define the role of NO in brachial artery flow-mediated vasodilation (FMD) in young, healthy humans. Brachial artery blood velocity and diameter were determined (ultrasound Doppler) in 8 volunteers (26 ± 1 year) before and after 5-minute forearm circulatory occlusion with and without intra-arterial infusion of the endothelial NO synthase inhibitor N(G)-monomethyl-L-arginine (L-NMMA; 0.48 mg/dL per minute). Control (CON) and L-NMMA trials were performed with the occlusion cuff placed in the traditional distal position, as well as proximal to the measurement site. FMD was significantly reduced, but not abolished, by L-NMMA in the distal cuff trial (8.9 ± 1.3%-6.0 ± 0.7%, CON versus L-NMMA; P=0.02), with no effect of L-NMMA on FMD with proximal cuff placement (10.6 ± 1.2%-12.4 ± 1.7%, CON versus L-NMMA; P=0.39). When the reduction in shear stimulus after L-NMMA was taken into account, no drug difference was observed for either distal (0.26 ± 0.02-0.23 ± 0.03, CON versus L-NMMA; P=0.40) or proximal (0.23 ± 0.08-0.23 ± 0.03, CON versus L-NMMA; P=0.89) FMD trials. These findings challenge the assertion that NO is obligatory for brachial artery FMD and call into question the sensitivity of this procedure for noninvasive determination of NO bioavailability in young, healthy humans.