A rapid preparative scale purification of erythrocyte free cholesterol has been developed for measurements of in vivo cholesterol synthesis from 2H2O. The quantity and purity of cholesterol obtained is suitable for combustion, zinc reduction of the water formed, and determination of deuterium isotopic content by gas isotope ratio mass spectrometry. The ability to detect and to quantitate a range of cholesterol synthesis rates is illustrated by measurements on young pigs receiving diets without and with added dietary cholesterol.