Auto-oxidation products of cholesterol may play a role in atherogenesis. In order to determine whether cholesterol or 25-hydroxycholesterol, a cholesterol auto-oxidation product, affected growth of vessel wall cells, sparse and confluent cultures of rabbit thoracic aorta smooth muscle cells and human umbilical vein endothelial cells were exposed to these compounds for 88 hr. The compounds were administered at 10⁻⁴, 10⁻⁵, 10⁻⁶ or 10⁻⁷ M in either ethanol or fetal bovine serum (FBS) vehicle. Cells were counted electronically, and the results were expressed as the percent growth in experimental vs control wells. Cholesterol did not inhibit cell growth under any experimental condition. 25-Hydroxycholesterol had the following effects: inhibited confluent smooth muscle cell growth at 10⁻⁴ M in ethanol vehicle only; inhibited sparse smooth muscle cell growth in a dose-related manner at 10⁻⁴, 10⁻⁵ and 10⁻⁶ M in ethanol vehicle, but in FBS vehicle inhibited at only 10⁻⁴ and 10⁻⁵ M; inhibited confluent human umbilical vein endothelial cells at 10⁻⁴ M in ethanol vehicle only; and inhibited sparse human umbilical vein endothelial cell growth at 10⁻⁴ and 10⁻⁵ M in ethanol vehicle only. Thus, rabbit aortic smooth muscle cell growth was more sensitive to inhibition by 25-hydroxycholesterol than human umbilical vein endothelial cell growth was.