The presence of cholestanol had never been reported in cultured cells, but in the present study cholestanol was detected both in human and rat skin fibroblasts. The present study has shown that the origin of cholestanol in these fibroblasts was derived from de novo synthesis in the cells rather than from the fetal calf serum used in the growth medium. The activity of 5 alpha-cholestan-3-one reductase which is the enzyme catalyzing the final step in the biosynthesis of cholestanol was measured by mass fragmentography using deuterium labeled 5 alpha-cholestan-3-one as a substrate. The activity of this enzyme was also detected in the microsomal fractions of rat cerebrum and cerebellum as well as in the liver. These results suggest that cholestanol can probably be synthesized in extra-hepatic tissues as well as in the liver.