Our principal objectives are to determine whether the prompt suppression of cholesterol synthesis, previously shown to occur after the administration of cholesterol to intact rats and almost certainly due to factors other than suppression of enzyme synthesis, can be produced in preparations of isolated liver; and to elucidate the mechanism of this suppression by experiments with cell-free preparations. The techniques to be used are intravenous injection of cholesterol in the form of lipoproteins into intact rats; perfusion of rat livers with hypercholesteremic blood; incubation of dispersed liver cells in media containing cholesterol in various forms; and incubation of homogenates and fractions derived from them, to test the hypothesis that the initial effect of cholesterol on its own synthesis is brought about by the removal or inactivation of a substance which stimulates the activity of HMG-CoA reductase. Although we believe that the importance of changes in enzyme concentration in the regulation of cholesterol synthesis is now firmly established, we have evidence which points strongly to the existence of another process that operates much more rapidly than is possible with that mechanism. Explanation of our results by cessation of enzyme synthesis would imply a half-life of HMG-CoA reductase of the order of 15-30 minutes, whereas the observed decay time of that enzyme is over 4 hours. Therefore, a vigorous search for other mechanisms is indicated.