The metabolism of [2,4-3H]5 alpha-cholest-8(14)-en-3 beta-ol-15-one (I) has been studied in Chinese hamster ovary (CHO-K1) cells which were maintained in a lipid-deficient medium. The incorporation of I into the cells was linear with respect to sterol concentration in the medium over the ranges of concentrations studied and was more than 3.5 times that of the uptake of cholesterol. The results of detailed chromatographic analyses of the lipids recovered from the cells after 6 h of incubation with [2,4-3H]I (0.5 microM or 6.0 microM) indicated that most of the 3H was associated with free I. Considerably lesser amounts of the 3H was associated with esters of I. No formation of [3H]cholesterol or [3H]cholesteryl esters (or other C27 monohydroxysterols) from labeled I was observed. The labeled material with the chromatographic behavior of the esters of I gave, after mild alkaline hydrolysis, the free 15-ketosterol which was characterized by the results of chromatographic and cocrystallization studies. Upon transfer of the CHO-K1 cells from a culture medium containing 8% newborn calf serum to the same medium containing 8% lipid-deficient newborn calf serum, increases in the levels of activity of cytosolic acetoacetyl-CoA thiolase and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase and of HMG-CoA reductase were observed. These increases were blocked by the addition of I at a concentration of 1.0 microM. I (1.0 microM) also caused a decrease in the levels of activity of the three enzymes in cells previously grown in medium containing lipid-deficient serum. These results demonstrate that I not only affects the enzymatic reduction of HMG-CoA but also the enzymatic formation of this key intermediate in cholesterol biosynthesis.