Dietary supplements containing L-arginine have been marketed with the purpose of increasing vasodilatation, and thus, blood and oxygen supply to the exercising muscle. The present study evaluated the acute effect of L-arginine supplementation on indicators of NO production, nitrite (NO2-) + nitrate (NO3-) (NOx), in healthy subjects. Plasma concentrations of asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) have also been addressed. Seventeen healthy males participated in a randomized, double-blind, placebo-controlled study. Blood samples were drawn from a left antecubital vein at baseline (T0). Afterwards, subjects were randomly submittedto 6 g of oral L-arginine supplementation (as L-arginine hydrochloride) or placebo (as corn starch); afterwards, the subjects remained at rest in supine position and blood samples were drawn again at 30 (T1), 60 (T2), 90 (T3) and 120 minutes (T4) after supplementation. To analyze NO production, NO3- was converted to NO2- by nitrate reductase, followed by the derivatization of NO2- with 2,3-diaminonaphthalene. NOx, ADMA and SDMA were analyzed using a high-performance liquid chromatography system and monitored with a fluorescence detector. Two-way ANOVA with repeated measures showed no significant changes in NOx concentrations on the L-arginine group as compared to placebo group at any of the fivetime points (T0: 17.6 ± 3.9 vs 14.6 ± 2.3 μmol/L; T1: 15.8 ± 2.4 vs 14.3 ± 1.7 μmol/L; T2: 16.8 ± 4.9 vs 13.7 ± 2.7 μmol/L; T3: 16.7 ± 3.9 vs 14.6 ± 2.1 μmol/L; T4: 15.1 ± 2.8 vs 13.5 ± 3.5 μmol/L). Furthermore, plasma levels of ADMA and SDMA were not statistically significant between the L-arginine and placebo groups at T0 (0.43 ± 0.19 vs 0.39 ± 0.15 μmol/L and 1.83 ± 1.13 vs 1.70 ± 0.62 μmol/L), respectively. In conclusion, acute L-arginine supplementation does not increase plasma concentration of NOx in healthy individuals with normal plasma concentrations of ADMA.