In the present study, we investigated the hypocholesterolemic effect of F-1394 ((1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocycloh exane-1-yl 3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino] propionate), a potent and selective inhibitor of acyl-CoA:cholesterol acyltransferase (ACAT), and the effect on cholesterol absorption via the gut in rats fed a 1% cholesterol diet. Single administration of F-1394 to the cholesterol diet-fed rats at the doses of 3-30 mg/kg, p.o. decreased the serum cholesterol levels by 16-54% 3 hr after the administration. The ACAT activity in the small intestinal mucosa of the rats given orally F-1394 (30 mg/kg) was significantly inhibited 3 hr after the administration. The hypocholesterolemic action of F-1394 had a faster onset than that of DL-melinamide or CL-277,083. The study by the dual isotope ratio method showed that F-1394 (30 mg/kg, p.o.) significantly suppressed the dietary cholesterol absorption. Furthermore, in the determination of cholesterol absorption by using 14C-cholesterol as the oral tracer, the administration of F-1394 (30 mg/kg, p.o.) 1 or 2 hr before or immediately after the application of the oral tracer significantly prevented the appearance of the radioactivity in the circulation by around 90%. These results indicate that oral administration of F-1394 inhibits the ACAT activity in the small intestinal mucosa and subsequently contributes much to the prevention of cholesterol absorption via the gut, resulting in the decrease in serum cholesterol levels in the cholesterol diet-fed rats. Furthermore, the effect of F-1394 appears immediately after its administration in contrast to that of DL-melinamide or CL-277,082.