To examine the validity of the deuterium (D) incorporation technique for measurement of human cholesterol synthesis rates, D uptake from D2O into cholesterol was compared to sterol balance in 13 subjects each under three controlled diet settings. Subjects (age 62 +/- 3.6 yr, body weight 74 +/- 4.0 kg, BMI 27 +/- 1.4) consumed weight maintenance diets enriched in either corn oil, beef tallow, or stick corn oil margarine over a 5-week period. During the final week of the study period, subjects were given 1.2 g/D2O per kg body water. D enrichment was measured in plasma water and total cholesterol over 24 h. Also, during the final week, dietary intake and fecal elimination rates of cholesterol were assessed over one 6-day period to calculate sterol balance. There was no significant difference (t = 0.858, P = 0.397) between D incorporation into cholesterol (1,183 +/- 92 mg/day) and sterol balance (1,316 +/- 125 mg/day). Among diets, net cholesterol biosynthesis measured by D incorporation agreed (r = 0.745, P = 0.0001) with values derived from sterol balance. The degree of association between methods was not influenced by the wide range of fatty acid composition of the diet fat. These data demonstrate the utility of the simple, non-restrictive deuterium incorporation method as a reliable means of determining cholesterol biosynthesis in free-living humans.