This study was undertaken to determine the mechanisms that regulate cholesterol synthesis in vivo and to quantitate the relative importance of the liver and extrahepatic tissues as sites for sterol synthesis. Rats were administered [3H]water intravenously and killed 1 hour later. The amount of [3H]water incorporated into digitonin-precipitable sterols was then measured in liver, whole blood, and the remaining tissues of the carcass. In control animals, killed at the mid-dark point of the light cycle, rates of [3H]water incorporation into sterols equaled 2290 and 103 nmol/hr per g, respectively, in the liver and carcass. Cholesterol feeding suppressed synthesis in the liver but not in the extrahepatic tissues, while fasting for 48 hr suppressed synthesis in both the liver and carcass. In fasted animals subjected to stress there was a 5-fold increase in hepatic synthesis but no change in synthesis by the extrahepatic tissues. Similarly, incorporation of [3H]water into sterols by the carcass was unaffected by light cycling while the liver showed a definite diurnal rhythm. In control rats, 34.5 mumol of [3H]water was incorporated into sterols by the whole animal per hour. Of this amount of sterol synthesis about 54% took place in the liver while the remaining amount occurred in the tissues of the carcass. With cholesterol feeding or fasting, or during the mid-light phase of the light cycle, synthesis in the extrahepatic tissues accounted for 69 to 90% of total body sterol synthesis.