Healthy young men were fed four diets for 2 weeks each providing natural fats containing palmitic acid (16 : 0) predominantly in the sn-1, 3 position of dietary TAG or containing 16 : 0 predominantly in the sn-2 position with low or high levels of linoleic acid (18 : 2n-6). Two treatments supplied 16 : 0 in the sn-1, 3 positions from palmstearin with low (3 % energy) or high (>7 % energy) 18 : 2n-6 and two treatments supplied 16 : 0 in the sn-2 position from lard with high or low levels of 18 : 2n-6. Diets contained 30–35 % energy as fat, 7–11 % energy as 16 : 0 and moderate levels of cholesterol. Fasting serum cholesterol and lipoprotein concentrations were measured. Cholesterol fractional synthesis rate (FSR) was determined by 2H incorporation. Diets providing 16 : 0 in the sn-2 position resulted in lower fasting serum total cholesterol (TC) and a lower TC:HDL ratio than diets providing 16 : 0 in the sn-1, 3 positions. Diets with high levels of 18 : 2n-6 significantly decreased the TC:HDL ratio, reaffirming the well-known cholesterol-reducing effect of 18 : 2n-6. A lower non-esterified cholesterol FSR was observed with low dietary levels of 18 : 2n-6. No differences between dietary treatments were found for serum HDL-cholesterol, LDL-cholesterol or TAG. It is concluded that dietary fats containing 16 : 0 in the sn-2 position may result in slightly lower fasting TC than diets providing 16 : 0 in the sn-1, 3 positions, while the level of n-6 polyunsaturated fat influences endogenous cholesterol synthesis.