Abstract-Endothelial dysfunction is often associated with a relative substrate deficiency of the endothelial nitric oxide synthase (eNOS) in spite of apparently high intracellular arginine concentrations. For a better understanding of the underlying pathophysiological mechanisms, we aimed to characterize the intracellular arginine sources of eNOS. Our previous studies in human endothelial EA.hy926 cells suggested the existence of two arginine pools: pool I can be depleted by extracellular lysine, whereas pool II is not freely exchangeable with the extracellular space, but accessible to eNOS. Key Words: endothelial nitric oxide synthase Ⅲ neutral amino acid transport Ⅲ system N Ⅲ intracellular arginine pool Ⅲ proteasome N O synthesized from arginine by endothelial nitric oxide synthase (eNOS) is a potent vasodilator and a critical modulator of blood flow and blood pressure. In addition, it mediates vasoprotective actions through inhibiting smooth muscle cell proliferation, platelet aggregation, and leukocyte adhesion. Under pathophysiological conditions associated with endothelial dysfunction, such as diabetes, hypertension, or hypercholesterolemia, supply of the substrate arginine seems to be limiting for NO synthesis. 1 This is in spite of intracellular arginine concentrations sufficiently high to saturate eNOS, a phenomenon termed the arginine paradox. 2 In order to understand this paradox, it seems important to elucidate the intracellular substrate sources for eNOS. Our previous studies in human endothelial EA.hy926 cells have demonstrated the existence of two arginine pools: pool I can be depleted by extracellular lysine through an exchange mechanism mediated by membrane transporters such as the cationic amino acid transporter 1 (CAT-1) or the system y ϩ L transporter 4F2hc/y ϩ LAT2. 3 Both transporters are expressed in endothelial cells and catalyze the exchange of cationic amino acids. 4,5 In addition, 4F2hc/y ϩ LAT2 mediates also the exchange of extracellular neutral amino acids against intracellular cationic amino acids. 6 In contrast to the arginine pool I, pool II is not freely exchangeable with extracellular lysine, but accessible to eNOS, thereby rendering eNOS independent of extracellular arginine. 3 The arginine paradox might therefore be explained by alterations in pool II or an impaired access of eNOS to pool II. Recent findings suggest that an increased production of the endogenous inhibitor asymmetrical dimethyl arginine (ADMA), derived from breakdown of proteins containing methylated arginine residues, might underlie the arginine paradox. 7-9 Plasma concentrations of ADMA are increased in patients suffering conditions associated with endothelial dysfunction. However, even the highest ADMA concentrations found in plasma from patients with renal failure are 5-to 10-fold lower than the plasma arginine concentrations. It is therefore tempting to speculate that ADMA might specifically accumulate in the arginine pool II of endothelial cells, thereby exerting a larger inhibitory action on eNOS than a...