Cholesteryl ester transfer protein (CETP) is a key protein involved in the reverse cholesterol transport pathway. The regulation of CETP by dietary fats is not clearly understood. Transgenic mice expressing human CETP under the control of its natural flanking region were fed low- or high-fat diets enriched in monounsaturated fatty acids (MUFAs) or saturated fatty acids in the presence or absence of cholesterol. Addition of cholesterol to the low-fat MUFA diet increased CETP activity and mRNA expression, whereas addition of cholesterol to the high-fat MUFA diet led to a decrease in CETP activity and mRNA expression. In SW 872 cells, oleic acid and cholesterol stimulated CETP gene expression when given alone. However, addition of fatty acids along with cholesterol interfered with the stimulatory effect of cholesterol on CETP gene regulation. Cholesterol-mediated stimulation of CETP involves the transcription factor liver X receptor alpha (LXRalpha). High-fat MUFA diets inhibited the expression of LXRalpha, and addition of cholesterol to the high-fat MUFA diet did not rescue LXRalpha expression. Therefore, we present evidence for the first time that inhibition of LXRalpha expression by a high-fat MUFA diet leads to inhibition of CETP stimulation by cholesterol.