The synthesis of cholesterol from labeled acetate and mevalonate by the liver and intestinal tract was investigated in germfree and conventional rats. When a low cholesterol diet was fed, the rates of in vitro synthesis from acetate by the liver, ileum and cecum of germfree rats were 13%, 11% and 25% of those of conventional rats, respectively. Cholesterol feeding markedly inhibited hepatic cholesterol synthesis from acetate in both germfree and conventional rats. Such inhibitions were released by additional cholestyramine feeding. The rate of hepatic cholesterol synthesis was greater and the liver cholesterol level was less after cholestyramine feeding in germfree rats than in conventional rats, suggesting an importance of bile acids in the regulation of cholesterol metabolism in the germfree rat. There was an inverse proportionality between the logarithmic rate of hepatic cholesterol synthesis and the liver cholesterol level in the germfree rat. Data indicate that endogenous cholesterol synthesis in the germfree rat may not be responsible for the high cholesterol level in plasma or liver and that the liver cholesterol level may play a major role in the regulation of hepatic cholesterogenesis in the germfree rat by a mechanism similar to that in the conventional rat.