The existence of a circadian rhythm in the rate of hepatic cholesterol synthesis in the rat has been demonstrated in vivo by measuring the conversion of both [1-(14)C]acetate and (3)H(2)O to cholesterol. By both methods there was observed a similar increase in the rate of hepatic cholesterol synthesis between the nadir at noon and the peak at midnight. Circadian changes in the rate of hepatic cholesterol synthesis measured in vivo with [1-(14)C]acetate were very similar to changes in the activity of hepatic microsomal HMG CoA reductase. Cholesterol synthesis in the jejunum and in the distal ileum was also shown to exhibit the same circadian rhythm in vivo but with smaller amplitude (1.6- and 1.3-fold, respectively). Rats trained to eat during a 4-hr period (9 am-1 pm) while housed under normal illumination showed changes in the timing of the circadian rhythm of cholesterol synthesis; in the liver the maximum rate of cholesterol synthesis occurred at 6 pm, 9 hr after the presentation of food, while the two sections of the intestine investigated exhibited a maximum synthetic response between noon and 6 pm. Results obtained in this study support the hypothesis that the major portion of the rise in HMG CoA reductase activity and the increase in overall rate of cholesterol synthesis in liver and intestine during the circadian rhythm are due to the ingestion of food. Under the limited feeding schedule (food access 9 am-1 pm), the rates of hepatic and intestinal synthesis of fatty acids from the injected acetate also showed a circadian rhythm with a peak at about 3 hr after presentation of food.