In order to assess the feasibility of using stable 13C as a tracer for studying cholesterol metabolism in vivo the 13C content of some commonly consumed foods and the stability of the 13C content of human serum cholesterol has been investigated. A procedure is described for the extraction and purification of cholesterol from food, tissues and serum suitable for 13C analysis by mass spectrometry. The method caused no significant isotope fractionation effects. Generally, there was very little difference between the carbon isotope composition of the diet, the cholesterol and the tissue from which it was derived. However, the difference in 13C composition between tissue and cholesterol of prawns from different parts of the world was considerable, indicating a possible peculiarity in the metabolism of cholesterol by this species. The 13C content of human serum cholesterol on a normal diet did not alter significantly with time, suggesting that stable isotope tracer techniques could be used to measure certain aspects of cholesterol metabolism in vivo.