L-arginine, as a precursor of NO synthesis, has attracted much scientific attention in recent years. Experimental mouse models suggest that L-arginine supplementation can retard, halt, or even reverse atherogenesis. In human studies, supplementation with L-arginine improved endothelium-dependent vasodilation. However, L-arginine levels are best interpreted in the context of levels of asymmetric dimethylarginine (ADMA), a competitive inhibitor of NO synthase. Thus, reference limits for circulating L-arginine and the L-arginine:ADMA ratio may help to determine the nutritional state of individuals at high cardiovascular risk in light of increased ADMA levels. We defined reference limits for plasma L-arginine in 1141 people and for the L-arginine:ADMA ratio in 1138 relatively healthy individuals from the Framingham Offspring Cohort. Plasma L-arginine and ADMA concentrations were determined by using a stable isotope-based LC-MS/MS method. The reference limits (2.5th and 97.5th percentiles) for plasma L-arginine were 41.0 μmol/L (95% CI = 39.5-42.5 μmol/L) and 114 μmol/L (95% CI = 112-115 μmol/L), whereas corresponding reference limits (2.5th and 97.5th percentiles) for the L-arginine:ADMA ratio were 74.3 μmol/L (95% CI = 71.1-77.3 μmol/L) and 225 μmol/L (95% CI = 222-228 μmol/L). Plasma L-arginine was positively associated with the estimated glomerular filtration rate (eGFR) and blood glucose levels, whereas the L-arginine:ADMA ratio was positively associated with eGFR and diastolic blood pressure but inversely associated with homocysteine and (log)C-reactive protein. We report reference levels for plasma L-arginine and for the L-arginine:ADMA ratio that may be helpful for evaluation of the effects of L-arginine supplementation in participants with an impaired L-arginine/NO pathway.