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For over a decade investigators have quantified cholesterol absorption by comparison of dietary intake and fecal excretion of isotopic cholesterol with that of beta-sitosterol as a "nonabsorbable" marker. However, beta-sitosterol might not be ideal due to its potential for absorption. We therefore carried out two studies to evaluate a new marker with less potential for absorption, [3H]beta-sitostanol. In the first study (Study I, n = 22), we compared absorption of [3H]beta-sitostanol and [14C]beta-sitosterol in a simultaneous dual-label continuous feeding ("phytosterol absorption") experiment. We observed a consistently higher ratio of [3H]beta-sitostanol/[14C]beta-sitosterol in the stool relative to diet on the first day of fecal collection (6.1% +/- 3.2% loss of [3H]beta-sitosterol, range 3-12%), but thereafter, the ratio in stool was similar to that in diet. In Study II (n = 23), we compared cholesterol absorption directly using [3H]beta-sitosterol and [14C]cholesterol, and, separately, [3H]beta-sitostanol and [14C]cholesterol. We found that mean absorption between the two methods was similar (45% +/- 11% versus 44% +/- 10%, respectively, P difference = 0.40), and the two methods correlated well with one another (r = 0.83) when samples from all available days were used. Variability between the two methods was greater in individuals who absorbed more than 40% of cholesterol. Cholesterol loss on day 2 estimated from use of beta-sitostanol as a nonabsorbable marker was predictive of absorption using ratios from days 4-6 (r = 0.80). These results suggest that, for the majority of subjects, beta-sitosterol is a valid nonabsorbable marker for cholesterol absorption.