We studied the effects of hypercholesterolemia on vascular responsiveness in different arteries isolated from rabbits: control groups of rabbits and groups receiving the atherogenic diet consisted of eight animals each. In the arteries, 16 weeks of cholesterol-rich (0.3%) diet evoked intimal lesions which were more pronounced than those noted after 8 weeks of hypercholesterolemia; the aortic arch was affected significantly more by the lesions than the abdominal aorta and the pulmonary artery. Segments of the arteries were mounted in organ chambers for isometric tension recording or for measurement of the endothelium-derived relaxant factor. Contractions caused by acetylcholine and prostaglandin F2 alpha were not altered by the hypercholesterolemia; those evoked by serotonin were moderately augmented only in the aortic arch of hypercholesterolemic rabbits. As the degree of intimal lesion formation increased, the contractions to norepinephrine and clonidine were progressively inhibited. The endothelium-independent relaxations to nitroglycerin were inhibited in only the most severely affected arteries; the endothelium-dependent relaxations to acetylcholine and adenosine triphosphate were progressively inhibited as the degree of fatty streak formation augmented. Thus, in the aortic arch, the relaxations to 3 X 10(-6) M acetylcholine, expressed as percent of the initial contraction, decreased from 86.7 +/- 3.3% in control tissues to 16.3 +/- 8.6% in the 16-week hypercholesterolemic vessels; in the abdominal aortas these relaxations averaged 93.5 +/- 2.2% in control vessels and 72.0 +/- 6.9% in the hypercholesterolemic tissues. The acetylcholine-induced release of endothelium-derived relaxant factor from the abdominal aorta was not significantly affected by the hypercholesterolemia. We conclude from these studies that in arteries obtained from hypercholesterolemic rabbits: the contractions caused by serotonergic mechanisms tend to be augmented, while those to alpha-adrenergic activation are decreased, the endothelium-independent relaxations are modified only in the more severely affected arteries, and the endothelium-dependent relaxations are progressively inhibited as the degree of fatty streak formation augments, probably because a step subsequent to the release of endothelium-derived relaxant factor is altered.