We have studied the in vivo inhibition of hepatic sterol biosynthesis by 1233A, a specific inhibitor of the enzyme 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase. Administration of the compound orally to mice resulted in a dose-dependent inhibition of [14C]acetate incorporation into sterols in liver, but did not exert any significant effect on [14C]mevalonate incorporation. The results indicate that the in vivo inhibition of sterol synthesis occurs only at pre-mevalonate enzymatic steps in the sterol biosynthetic pathway, thus being compatible with the presumed site of inhibition, HMG-CoA synthase. Moreover, owing to irreversible inactivation of the enzyme by 1233A, it was possible to detect in vivo effect on the enzyme by assays of its activity in cell-free extracts from livers; the drug-treatment also resulted in a similarly dose-dependent inhibition of HMG-CoA synthase activity. In contrast, acetoacetyl-CoA thiolase and HMG-CoA reductase, the enzymes also responsible for mevalonate synthesis in the pathway, did not show any significant change in activity. These results clearly demonstrate that the inhibition of hepatic sterol synthesis caused by 1233A is indeed due to selective inhibition of HMG-CoA synthase in the tissues.