Bioassay studies suggest that impaired endothelium-dependent relaxation in atherosclerotic arteries is due to a reduced release of biologically active endothelium-derived relaxing factor (EDRF). We tested the hypothesis that endothelial dysfunction is caused by deficiency of the EDRF precursor L-arginine. Aortae from normal and cholesterol-fed (1%, 4 months) rabbits were excised and incubated for 1 h with 5 m M L-arginine. Pretreatment with L-arginine had no effect on the relaxation to acetylchohne in normal vessels and was without effect on the impaired response of atherosclerotic arteries to acetylchohne. This finding suggests that L-arginine deficiency is unlikely the underlying cause of impaired endothelium-dependent relaxation in the aorta of cholesterol-fed rabbits.