Endothelial function is impaired in hypercholesterolemia and atherosclerosis, which is probably due to reduced biological activity of endothelium-derived nitric oxide (NO). NO is synthesized in functionally intact endothelium by oxidation of the terminal guanidino nitrogen atom(s) of the amino acid precursor, l-arginine. We applied stable isotope dilution techniques and gas chromatographic–mass spectrometric approaches to investigate metabolism of l-[guanidino- 15 N 2 ]-arginine to 15 N-labeled nitrate in hypercholesterolemic rabbits and controls. After 4 weeks on control or 1% cholesterol-enriched diet, rabbits received 267±6μmol of l-[guanidino- 15 N 2 ]-arginine/kg of body weight via gastric cannulation. 15 N-isotope content of l-arginine in plasma and in platelet lysates increased 2h later in both groups, and almost returned to baseline until 24h. 15 N-isotope content of plasma nitrite and nitrate also increased in both groups at 2h, and had almost returned to natural content 24h later. 15 N-isotope content of urinary nitrate was significantly increased in control animals in urines collected from 0 to 12, 12 to 24, and had returned to baseline in the urine sample collected from 24 to 48h. In the cholesterol group only a slight, insignificant elevation of 15 N-isotope content was observed for urinary nitrate. The extent of conversion of l-[guanidino- 15 N 2 ]-arginine to 15 N-labeled nitrate was strongly and inversely correlated to plasma concentration of the endogenous NO synthase inhibitor, asymmetric dimethylarginine (ADMA), which was elevated in cholesterol-fed rabbits (R=0.77; p<0.05). Our data show that baseline NO synthase turnover rate is reduced in rabbits during early hypercholesterolemia. Our study gives evidence that the mechanism of the impaired conversion of l-[guanidino- 15 N 2 ]-arginine to 15 N-labeled nitrate most likely involves inhibition of NO synthase by ADMA, which is present in elevated concentrations in hypercholesterolemia.