Background: The assessment of altered nitric oxide (NO) availability is of potentially important diagnostic and prognostic significance. The present study is aimed to investigate the effect of L-arginine (as a natural NO donor) supplementation on NO metabolite in a rabbit model of hypercholesterolemia to find a reliable marker for endothelial NO production. Methods: White male rabbits (n = 30) randomly assigned to 2 groups. Rabbits were fed 1% high-cholesterol diet (HC group, n = 15), or HC diet with oral L-arginine (3% in drinking water) (HC + L-arginine group, n = 15) for 4 weeks. The serum levels of lipids, L-arginine, total NO metabolites (NOx), NITRITE and nitrate were measured before and after the study. Results: In this study, Larginine supplementation led to a significant increased plasma level of L-arginine. The serum level of NITRITE was significantly higher in L-arginine treated group while serum level of nitrate and NOx was significantly lower than HC group. Conclusion: As the result of our study showed, NITRITE is a useful marker of endogenous endothelial NO production and although frequently used, neither nitrate nor NOx are reliable markers of acute changes in endothelial NO synthase activity.