Asymmetrical dimethylarginine (ADMA) is an endogenous nitric oxide synthase inhibitor. It is formed by protein arginineN-methyltransferases (PRMTs), which utilizeS-adenosylmethionine as methyl group donor. ADMA plasma concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and producing proatherogenic changes of endothelial cell function. Four different isoforms of human PRMTs have been identified. Because the release of ADMA from human endothelial cells is increased in the presence of native or oxidized LDL cholesterol, we investigated the potential involvement of PRMT activity and gene expression in this effect. We found that the production of ADMA by human endothelial cells is upregulated in the presence of methionine or homocysteine and inhibited by either of the methyltransferase inhibitorsS-adenosylhomocysteine, adenosine dialdehyde, or cycloleucine. This effect is specific for ADMA but not symmetrical dimethylarginine. The upregulation of ADMA release by native and oxidized LDL is abolished byS-adenosylhomocysteine and by the antioxidant pyrrollidine dithiocarbamate. Furthermore, a methyl-14C label is transferred fromS-adenosylmethionine to ADMA but not symmetrical dimethylarginine, in human endothelial cells. The expression of PRMTs is upregulated in the presence of native or oxidized LDL. Our data suggest that the production of ADMA by human endothelial cells is regulated byS-adenosylmethionine–dependent methyltransferases. This activity is upregulated by LDL cholesterol, which may be due in part to the enhanced gene expression of PRMTs. In concentrations reached by stimulation of methyltransferases (5 to 50 &mgr;mol/L), ADMA significantly inhibited the formation of15N-nitrite from l-[guanidino-15N2]arginine. These findings suggest a novel mechanism by which ADMA concentration is elevated in hypercholesterolemia, leading to endothelial dysfunction and atherosclerosis.(Circ Res. 2000;87:99-105.)