The effect of conjugated linoleic acid (CLA) isomers esterified in diacylglycerol (DAG)-rich oil on lipid metabolism was investigated. Since dietary DAG has been known to induce the regression of atherosclerosis, CLA-DAG and olive-DAG oils containing similar levels of DAG (51.4~54.2%) were synthesized from olive oil. Hyperlipidemic C57BL/6J mice were then fed high-fat high-cholesterol diets supplemented with these oils (5% each) for 7 wk. The CLA-DAG diet containing 2.1% CLA isomers (0.78% c9,t11-CLA; 1.18% t10,c12-CLA) remarkably increased the levels of total plasma cholesterol and glutamic oxaloacetic transaminase (GOT) along with hepatic cholesterol and triacylglycerol (TAG) contents. Furthermore, the CLA-DAG diet inhibited fat uptake into adipose tissue whereas fat deposition (especially in the liver) was increased, resulting in the development of fatty livers. Hepatic fatty acid composition in the CLA-DAG mice was different from that of the olive-DAG mice, showing higher ratios of C16:1/C16:0 and C18:1/C18:0 in the liver. The activity of hepatic acyl-CoA:cholesterol acyltransferase (ACAT) was higher in CLA-DAG mice while plasma lecithin:cholesterol acyltransferase (LCAT) activity and the ferric reducing ability of plasma (FRAP) were lower in CLA-DAG mice compared to the olive-DAG animals. Results of the present study suggest that CLA incorporation into DAG oil could induce atherosclerosis in mice.