The effect of dietary fat sources with different degrees of unsaturation and double bond positioning on lipid metabolism was studied in growing chicks. Four-week-old chicks were given semi-purified diets containing 6% palm oil (PLO), 6% safflower oil (SFO), 3% safflower oil and 3% linseed oil (SFO + LNO), 6% linseed oil (LNO), or 3% linseed oil and 3% fish oil (LNO + FO) with 0.5% cholesterol supplementation. Cholesterol ester content in the liver and serum of chicks fed PLO diet was significantly higher than that of other diet groups. Liver triacylglycerol and free cholesterol contents were significantly decreased in chicks fed a diet containing n-3 fatty acids (i.e., linseed oil or fish oil). Serum triacylglycerol level was also decreased by feeding the LNO or LNO + FO diet. The activity of serum lecithin-cholesterol acyltransferase was not affected by dietary treatments. Fecal neutral steroid excretion was significantly increased in the LNO+FO diet group as compared with the SFO + LNO or LNO diet groups. With the increase in linseed oil levels, the levels of C18:2n6 and C20:4n6 fatty acids in tissue lipids decreased, but C18:3n3 and C20:5n3 were gradually increased. The levels of longer chain n-3 fatty acids (i.e., C20:5n3 or C22:6n3) in chicks fed a diet containing fish oil (LNO + FO diet) were significantly increased compared to those fed linseed oil with a corresponding level. These results demonstrated that dietary fat enriched with alpha-linolenic acid and longer chain n-3 fatty acids have stronger effects on lowering serum lipid levels than dietary fat composed of either saturated, or n-6 fatty acids, but both n-3 fatty acids sources show differing effects on the deposition of longer chain n-3 fatty acids into tissue lipids.