We studied the effect of dietary cholesterol on tissue cholesterol synthesis in 10 genetically lean pigs using the 2H2O incorporation method. Five pigs were fed a diet containing 0% cholesterol (controls) and five were fed a diet containing 0.5% cholesterol (cholesterol-fed) from d 1 to d 54 of age. From d 30 to d 54, they received 2H2O and cholesterol synthesis was determined by measuring deuterium enrichment in erythrocyte cholesterol. All pigs were killed on d 54 and liver, ileum, jejunum, kidney, adipose tissue, muscle, and cerebrum were collected for measurement of free and esterified cholesterol and of the deuterium content of tissue free cholesterol. In vivo cholesterol synthesis was inhibited by dietary cholesterol as reflected by a decrease in fractional synthesis rate from 16.8 +/- 1.7%/d in controls to 2.3 +/- 1.1%/d in cholesterol-fed pigs. Cholesterol feeding raised cholesterol ester concentration in plasma, liver, and other tissues. Deuterium enrichment of free cholesterol in tissues, such as liver, was reduced from 11.98 +/- 2.82 milli atom percent excess in controls to 1.83 +/- 1.14 in cholesterol-fed pigs. Greater cholesterol intake did not inhibit cholesterol synthesis in the cerebrum. Deuterium enrichment levels of cholesterol in plasma and liver were similar to those in other tissues except for brain, suggesting an equilibration of cholesterol between the liver and most extrahepatic tissues. The control of cholesterol synthesis in brain appears to be independent of that in liver and other tissues of the body.