The present study was designed to study the mechanisms by which dietary conjugated linoleic acids (CLA) decrease serum cholesterol. Hamsters were fed a semi-synthetic diet containing 1 g cholesterol/kg diet with or without supplementation with 20 g linoleic acid (LA) and 20 g CLA/kg diet. After 8 weeks, serum fasting total cholesterol (TC) and triacylglycerol (TG) were significantly lower in the LA-supplemented and CLA-supplemented groups compared with those of the control (CTL) hamsters. In contrast to LA, CLA significantly lowered hepatic cholesterol but it increased the level of adipose tissue cholesterol, suggesting that the hypocholesterolaemic mechanism of CLA is different from that of LA. CLA decreased the activity of intestinal acyl CoA:cholesterol acyltransferase (ACAT) whereas LA had no effect on this enzyme. Consequently, CLA supplementation increased the faecal excretion of total neutral sterols, but it had no or little effect on the faecal acidic sterols. If the ACAT is associated with cholesterol absorption, the part of mechanisms by which CLA decreases serum cholesterol may involve down-regulation of intestinal ACAT activity.