The rate of whole bodyb cholesterol synthesis was measured in male Sprague-Dawley rats fed either a standard chow, cereal-based diet or a semi-synthetic purified diet consisting of casein, sucrose and lard. The purified diet significantly decreased daily fecal excretion of neutral and acidic sterols, the specific acitvity of hepatic 3-hydroxy-3-methylglutaryl coenzyme A reductase, the bile acid pool size, and total daily cholesterol synthesis in the rat, while increasing plasma cholesterol concentrations and the total body content of cholesterol. The increased body content of cholesterol occurred primarily in muscle and connective tissue and not in the liver. The data demonstrate the importance of quantitating the net tissue accumulatin of cholesterol for accurate measurement of daily sterol synthesis in growing animals when sterol balance measurements are used. Tissue accumulation accounted for 7% of total daily cholesterol synthesis in rats fed the cereal diet, and 20% of daily synthesis in animals fed the purified diet.