24S,25-Epoxycholesterol is formed in a shunt of the mevalonate pathway that produces cholesterol. It is one of the most potent known activators of the liver X receptors and can inhibit sterol regulatory element-binding protein processing. Until recently analysis of 24S,25-epoxycholesterol at high sensitivity has been precluded by its thermal lability and lack of a strong chromophore. Here we report on the analysis of 24S,25-epoxycholesterol in rodent brain where its level was determined to be of the order of 0.4-1.4 mu g/g wet weight in both adult mouse and rat. For comparison the level of 24S-hydroxycholesterol in brain of both rodents was of the order of 20 mu g/g, while that of cholesterol in mouse was 10-20 mg/g. By exploiting knockout mice for the enzyme oxysterol 7 alpha-hydroxylase (Cyp7b1) we show that this enzymes is important for the subsequent metabolism of the 24S,25-epoxide. (C) 2014 The Authors. Published by Elsevier Inc.