This study was undertaken to determine the rates of sterol synthesis and uptake in the major organs of the female rat in vivo. At the mid-dark phase of the light cycle, control animals, animals in which hepatic sterol synthesis had been selectively inhibited by chylomicron infusion and animals in which the small intestine and liver had been surgically removed, were administered [(3)H]water, and the content of (3)H-labeled digitonin-precipitable sterols ([(3)H]-DPS) in different organs was measured 1 hr later. In control animals, the highest content of [(3)H]DPS was found in the liver (2279 nmol/hr per g), adrenal gland (1222), ovary (791), and small bowel (529): the content of newly synthesized sterols was much lower in 13 other tissues. By selectively inhibiting sterol synthesis in the liver or by surgically removing the small intestine and liver, it was determined that of the total amount of [(3)H]DPS synthesized in the whole animal about 50% had occurred in the liver, 24% in the small bowel, 8% in the skin, and 18% in the remaining tissues of the carcass combined. By analyzing the relationship between the content of [(3)H]DPS in blood and in each organ, it was further possible to determine how much [(3)H]DPS was synthesized and how much was taken up from the blood in each tissue. The highest rate of uptake was found in the adrenal gland where only 4% of the tissue content of [(3)H]DPS came from local synthesis. Low rates of synthesis relative to the rates of uptake, were also found in the spleen (6%), lung (17%), and kidney (26%). In contrast, in other organs there was little uptake of [(3)H]DPS from blood so that >75% of the [(3)H]DPS present in brain and muscle, for example, was due to local synthesis. Lowering the circulating levels of plasma cholesterol markedly increased the synthesis of [(3)H]DPS in tissues like adrenal gland, spleen, and kidney that were dependent upon plasma cholesterol as the major source of tissue sterols, but not in tissues such as muscle and brain. These studies have quantitated the importance of each major organ to total body synthesis and have delineated the rates of movement of [(3)H]DPS between major tissue compartments of the rat.-Turley, S. D., J. M. Andersen, and J. M. Dietschy. Rates of sterol synthesis and uptake in the major organs of the rat in vivo.