The purpose of this study was to determine if the nitric oxide synthase (NOS) inhibitor N^G-nitro-L-arginine methyl ester (L-NAME) demonstrates significant muscarinic receptor antagonism during methacholine (MCH) stimulated sweating in human forearm skin. Three intradermal microdialysis probes were placed in the skin of 8 healthy adults (4 men and 4 women). MCH in the range of 0.033 mM to 243 mM in 9 steps was perfused through a microdialysis probe with and without the presence of the NOS inhibitor L-NAME (10 mM) or L-arginine analogue N^G-monomethyl-L-arginine (L-NMMA, 10 mM). Local sweat rate (SR) and skin blood flow (laser Doppler velocimetry) were measured directly over each microdialysis probe. We observed similar resting SR at MCH only, MCH & L-NAME, and MCH & L-NMMA sites averaging 0.175 ± 0.029, 0.186 ±0.034, and 0.139 ± 0.027 mg/min/cm², respectively. Peak SR (0.46 ± 0.11, 0.56 ± 0.16, and 0.53 ± 0.16. mg/min/cm²) was also similar among all three sites. MCH produced a sigmoid shape dose response curves and 50 % of the maximal attainable response (EC₅₀) (0.42 ± 0.14 mM for MCH only) was shifted rightward shift in the presence of L-NAME or L-NMMA (2.88 ± 0.79 and 3.91 ± 1.14 mM, respectively, p<0.05). These results indicate that NO acts to augment MCH-stimulated sweat gland function in human skin. In addition, L-NAME consistently blunted the MCH-induce vasodilation while L-NMMA did not. These data support the hypothesis that muscarinic-induced dilation in cutaneous blood vessels is not mediated by nitric oxide production and that the role of L-NAME in attenuating acetylcholine-induced vasodilation may be due to its potential to act as a muscarinic receptor antagonist.