Cholesterol-fed rabbits were used to study the anti-atherosclerotic effect of (1S,2S)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]cyclohexane-1-yl 3-[(4R)-N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate (F-1394), an acyl-CoA:cholesterol acyltransferase (ACAT) inhibitor. To test its effect on the development of atherosclerosis, rabbits were fed a high-cholesterol diet (HCD) for 6 weeks, followed by regular chow (RC) for 12 weeks plus 0 or 100 mg/kg per day F-1394. Serum total cholesterol (TC) rose to approximately 2000 mg/dl on HCD and then declined gradually after the change in diet in both groups. F-1394 significantly reduced the extent of the atherosclerotic lesions and the total and esterified cholesterol contents of the aorta (by 57,38, and 59%, respectively), without affecting the serum TC level. To clarify whether F-1394 accelerates the regression of preexisting atherosclerosis, rabbits were fed HCD for the first 6 weeks and then RC for the next 6 weeks. Then, the rabbits were given 0 or 100 x 2 mg/kg per day F-1394 for another 12 weeks while on RC. F-1394 significantly reduced the extent of the atherosclerotic lesions and the total and esterified cholesterol content in the aorta (by 31, 31, and 43%, respectively), without affecting the serum TC level. These results demonstrate that F-1394 both prevents the formation of atherosclerosis and accelerates its regression without affecting the serum TC level, indicating that F-1394 acts directly on the arterial wall.