This study was aimed to examine the effect of L-arginine (ARG) exposure on the disposition of asymmetric dimethylarginine (ADMA) in human endothelial cells. Although the role of ADMA as an inhibitor of endothelial nitric oxide synthase (eNOS) is well-recognized, cellular interactions between ARG and ADMA are not well-characterized. EA.hy926 human vascular endothelial cells were exposed to 15N4-ARG, and the concentrations of 15N4-ARG and ADMA in the cell lysate and incubation medium were determined by a liquid chromatography-electrospray tandem mass spectrometry (LC-MS/MS) assay. Nitric oxide (NO) production was estimated by utilizing cumulative nitrite concentration via a fluorometric assay. Cells incubated with 15N4-ARG exhibited enhanced nitrite production as well as 15N4-ARG cellular uptake. These changes were accompanied by a decrease in cellular ADMA level and increase in extracellular ADMA level, indicating an efflux of endogenous ADMA from the cell. The time courses of ADMA efflux as well as nitrite accumulation in parallel with 15N4-ARG uptake were characterized. Following preincubation with 15N4-ARG and D7-ADMA, the efflux of cellular 15N4-ARG and D7-ADMA was significantly stimulated by high concentrations of ARG or ADMA in the incubation medium, demonstrating trans-stimulated cellular transport of these two amino acids. D7-ADMA metabolism was inhibited in the presence of added ARG. These results demonstrated that in addition to an interaction at the level of eNOS, ARG and ADMA may mutually influence their cellular availability via transport and metabolic interactions.