Intestinal cholesterol esterification by the enzyme acyl-CoA:cholesterol acyltransferase (ACAT) is a presumed prerequisite for cholesterol absorption. We evaluated the effect of a potent, poorly absorbed ACAT inhibitor (DuP 128: N'-(2,4-difluorophenyl)-N-[5-(4,5-diphenyl-1H-imidazol-2-ylthio)pe ntyl]- N-heptylurea) on cholesterol absorption in a randomized trial. Thirty subjects received DuP 128 for 7 weeks, 10 each at 900 mg per day, 1800 mg per day, and 3600 mg per day; six subjects received placebo; and nine subjects received 1 gm neomycin twice a day. Cholesterol absorption determinations used a continuous dual isotope 14C-cholesterol and 3H-beta sitosterol method. DuP 128 (pooled doses) induced at 14.4% +/- 11.4% reduction in cholesterol absorption (p < 0.05 versus placebo): 17.6% +/- 8.4% at 900 mg, 9.1% +/- 11.4% at 1800 mg, and 17.1% +/- 12.9% at 3600 mg. Neomycin induced a 26.4% +/- 10.7% reduction (p < 0.01). After 6 weeks, neomycin reduced serum total and low-density lipoprotein cholesterol by 22.4% +/- 9.2% and 24.0% +/- 11.6%, respectively (p < 0.01 versus placebo). DuP 128 induced reductions of 3.9% +/- 11% (difference not significant) and 4.95% +/- 14.3% (p = 0.05). ACAT inhibitors limit cholesterol absorption in humans; however, the magnitude of the effect, as exemplified by DuP 128, is small.