A series of five experiments was conducted to determine the effect of pectin, gum arabic and agar (5%) on cholesterol absorption, biosynthesis and turnover in rats. In the study of cholesterol absorption, a tracer dose of labeled cholesterol was included in the last meal. The rats were killed 12 hours later. The proportion of the labeled cholesterol recovered in the whole body was used as an estimation of the efficiency of absorption of dietary cholesterol. Cholesterol biosynthesis was estimated by determining the activity of labeled digitonin-precipitable sterols biosynthesized from labeled glucose which was included in a test meal. In turnover studies, rats were injected intravenously with labeled cholesterol using serum as a vehicle, and the activity of labeled cholesterol in tissues was determined after various time intervals. All three complex carbohydrates decreased cholesterol absorption and pectin had the greatest effect. Pectin and gum arabic increased cholesterol biosynthesis in rats fed a cholesterol-containing diet, but had no effect in a cholesterol-free diet. Pectin slightly increased the turnover of cholesterol, but gum arabic and agar had no effect. This work supports the hypothesis that pectin lowers cholesterol levels by interfering with cholesterol absorption and by increasing cholesterol turnover. The study also suggests that complex carbohydrates differ in their effects on cholesterol metabolism. The reason for these differences remains to be determined.