It is commonly believed that the activity of NO synthase (NOS) solely controls NO production from its substrates, l-Arg and O₂. The Michaelis-Menten constant (K _m) of NOS forl-Arg is in the micromolar range; cellular levels of l-Arg are much higher. However, evidence strongly suggests that cellular supply of l-Arg may become limiting and lead to reduced NO and increased superoxide anion () formation, promoting cardiovascular dysfunction. Uptake ofl-Arg into cells occurs primarily (∼85%) through the actions of a Na⁺-independent, carrier-mediated transporter (system y⁺). We have examined the effects of NOS agonists (substance P, bradykinin, and ACh) and NO donors (S-nitroso-N-acetyl-penicillamine and dipropylenetriamine NONOate) on transport ofl-Arg into bovine aortic endothelial cells (BAEC). Our results demonstrate that NOS agonists increase y⁺ transporter activity. A rapidly acting NO donor initially increasesl-Arg uptake; however, after longer exposure, l-Arg uptake is suppressed. Exposure of BAEC withoutl-Arg to substance P and a Ca²⁺ ionophore (A-23187) increased formation, which was blocked with concurrent presence ofl-Arg or the NOS antagonistN ^ω-nitro-l-arginine methyl ester. We conclude that factors including NO itself control y⁺ transport function and the production of NO and .