Cholestanol is the 5α-saturated analog of cholesterol and its accumulation is a hallmark of cereberotendinous xanthomatosis (CTX), a slowly progressive disease due to loss-of-function genetic changes in sterol 27-hydroxylase or CYP27A1. In CTX, cholestanol is accumulated in different tissues, especially tendons and brain, thus leading to neurological, non-neurological, and ocular manifestations. The biochemical pathways of cholestanol accumulation in the brain are well established. However, it is not yet fully understood how cholestanol is eliminated from different tissues. Accordingly, this chapter describes studies aimed at the identification of the enzymes involved in cholestanol elimination from the brain and retina. These studies suggest that in the brain, cholestanol could be an endogenous substrate for both CYP27A1 and CYP46A1 and that in the brain cerebellum, cholestanol is mainly metabolized by CYP46A1. Similarly, CYP27A1 and CYP46A1 are important for cholestanol elimination from the retina, although in mice, cholestanol does not seem to accumulate in the retina when CYP27A1 is absent. Since CYP46A1 activity in the brain and retina could be increased pharmacologically by administration of low dose anti-HIV drug efavirenz, CYP46A1 should be considered as a potential therapeutic target for CTX treatment in patients who are not responsive to the standard chenodeoxycholic acid therapy.