4β-hydroxy Cholesterol, a major oxysterol found in human circulation, is formed from cholesterol by cytochrome P450 (CYP)3A4 and CYP3A5. It has an unusually long half-life in plasma (~60 hours) as a result of slow elimination, particularly due to a slow rate of 7α-hydroxylation, which is the rate-limiting step for further conversion into bile acids. This metabolite has been suggested as a marker for CYP3A4/5 activity.