The objective of this study was to measure the response of cholesterol biosynthesis in subjects to three different amounts of dietary cholesterol: 50 (low), 350 (medium), and 650 (high) mg cholesterol per 2800 kcal. Individuals with low (n = 7), normal (n = 12), and elevated (n = 11) plasma cholesterol concentrations consumed in random order solid-food test diets (15%, 55%, and 30% of energy as protein, carbohydrate, and fat, respectively) at each dietary cholesterol level. The three diets were consumed for 4 weeks each, and each dietary phase was separated by a 4-week washout period. During the final week of each diet, 0.7 g D2O was given per kilogram of body water and deuterium incorporation into the erythrocyte cholesterol pool was measured for 24 hours. Urinary mevalonate levels were also determined in samples obtained during two consecutive 24-hour periods. Both techniques provided measurements of whole-body cholesterol biosynthesis. In all subjects the cholesterol synthesis rate as measured by deuterium incorporation was significantly lower (P < .05) after the transition from low- to medium- and low- to high-cholesterol diets. Urinary mevalonate excretion decreased after the change from the medium- to high- (P < .05) and low- to high- (P < .01) cholesterol diets. Although correspondence between the two methods was poor, they both indicated some suppression of cholesterol synthesis by dietary cholesterol. The response of cholesterogenesis to different amounts of dietary cholesterol was related to the rate of synthesis under depressed conditions of the low-cholesterol diet. These findings indicate modest downregulation of synthesis in response to dietary cholesterol in humans, independent of plasma cholesterol levels.