Barley oil was extracted with hexane from the grain of a high oil waxy hull-less barley. Twelve male broiler chicks were fed corn-based diets with either 10% barley oil, 10% corn oil or 10% margarine ad libitum for ten days. Total plasma cholesterol concentration of the chicks fed barley oil was 34% lower (p < 0.05) than that of the chicks fed margarine. Plasma low density lipoprotein cholesterol concentration of chicks fed barley oil was 53% and 59% lower (p < 0.05) than those of chicks fed corn oil and margarine, respectively. Plasma high density lipoprotein cholesterol and triglyceride concentration of the barley oil group were similar to those of the margarine but higher (p < 0.05) than those of the corn oil group. Chicks fed the barley oil gained more (p < 0.05) body weight than those fed the corn oil and margarine. Barley oil had an effect in suppression of TC and LDLC in chicks compared to margarine. Barley oil suppressed LDLC but not HDLC in chicks compared to corn oil. A greater weight gain of the chicks fed barley oil suggested that these chicks had normally functioning digestion and absorption. alpha-Tocotrienol and gamma-tocotrienol content of the barley oil were 24 and 17 times greater, respectively, than those observed in the corn oil, while the same fractions were not detectable in the margarine. Polyunsaturated fatty acid content of the barley oil was more than threefold that of margarine. These data suggest that alpha-tocotrienol and polyunsaturated fatty acids are hypocholesterolemic components in barley oil.