Young male Sprague-Dawley rats were fed diets containing 10% fat with P/S and n-6/n-3 ratios of 1.2 and 5.0, respectively, and differing levels of dietary cholesterol (0.05 to 1.0% by weight) for 3 weeks, following which the tissue lipid concentration, fatty acid composition, desaturation of linoleic acid, and eicosanoid production were examined. The composition of dietary fat was that recommended for the prevention of atherosclerosis. Dietary cholesterol did not largely influence the concentration of serum cholesterol, but it markedly increased liver cholesterol in a dose-dependent manner. The delta 6-desaturase activity in liver microsomes, linoleate desaturation index in liver phosphatidylcholine, and the production of prostaglandin by the aorta and thromboxane A2 by platelets all decreased to a similar extent in relation to dietary cholesterol above 0.2%. Thus, dietary cholesterol influenced various lipid parameters characteristically even when dietary fat with a fatty acid composition desirable for the prevention of atherosclerosis was consumed simultaneously. The dietary cholesterol level of 0.2% was the point of maximum influence in rats upon the indices examined.