Cholesterol and its diagenetic congeners have been detected in a wide range of human and animal bones dating to the Mesolithic period. Using isotope ratio monitoring-gas chromatography/mass spectrometry (irm-GC/MS), it is possible to determine accurately compound specific δ13C values of preserved cholesterol with high precision (± 0·4 per mil). The δ13C value of cholesterol provides information complementary to that derived from collagen and apatite stable isotopes for use in palaeodietary studies. Potential advantages of using cholesterol are that: (1) the δ13C value will be biased towards the original isotopic composition of the carbohydrates and fats in the diet, (2) its δ13C value will retain isotopic integrity, i.e., will not be diagenetically altered, provided its carbon skeleton is characterized, and (3) that its relatively rapid rate of turnover provides a means of investigating carbon cycling on a shorter time scale than collagen. Laboratory animal feeding experiments have shown that cholesterol faithfully derives its isotopic signature from the diet and has a faster turnover rate than collagen. This paper reviews aspects of the methodologies employed in recording δ13C values from cholesterol preserved in skeletal materials and presents new results obtained from modern and archaeological humans, and studies of experimental animals.