Percent cholesterol absorption was measured in 94 normal subjects aged 17- 80 years while consuming diets generally low in cholesterol (mean intake = 226 +/- 126 mg/day). A new dual stable isotope method was used where a cholesterol tracer containing 6 extra mass units was given intravenously and another tracer with 5 extra mass units was given orally during a standard test meal. The ratio of tracers in plasma was determined by negative ion mass spectrometry of pentafluorobenzoyl sterol esters. Absorption values ranged widely from 29.0% to 80.1% with mean 56.2 +/- 12.1 (SD) %. Cholesterol absorption was significantly increased in African-Americans (63.4 +/- 11.8% vs. 55.1 +/- 11.9%, P = 0.027) but was similar for women (53.3 +/- 11.9%) and men (57.6 +/- 12.1%). It was not related to plasma lipoproteins, age, apoE3/E3 or E3/E4 genotype, or chronic dietary intake of energy, fat, or cholesterol quantitated from 7- day food records. However, dietary cholesterol intake was positively related to plasma cholesterol (P = 0.036) and triglycerides (P = 0.026). The milligram amount of dietary cholesterol absorbed (but not percent absorption) was positively correlated with fasting plasma insulin (r = 0.525, P < 0.0001), C-peptide (r = 0.367, P = 0.0003) and glucagon (r = 0.421, P < 0.0001) independent of gender, body fat percent and age.The efficiency of intestinal cholesterol absorption and the milligram amount of dietary cholesterol absorbed were not related to plasma cholesterol or LDL cholesterol in individuals consuming a low-cholesterol low-fat diet. The dominant factor determining dietary cholesterol absorption was intake rather than absorption efficiency. Dietary cholesterol and fat were strongly and independently related to hormonal measures of insulin resistance.-Bosner, M. S., L. G. Lange, W. F. Stenson, and R. E. Ostlund, Jr. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.