Alteration of cholesterol (Ch) homeostasis is involved in the pathogenesis of neurodegeneration. Astrocyte-derived lecithin cholesterol acyltransferase (LCAT) was recently shown capable of esterifying Ch on ApoE-lipoproteins. In order to prevent Ch excess in neurons, it can be converted in 24S-hydroxycholesterol (24-OH Ch). Since this cerebrosterol can cross the brain blood barrier, 24-OH Ch is thought to be a major form of Ch for egress from the brain. 24-OH Ch is known to be produced almost exclusively in the brain, and was so far only suggested to be LCAT substrate in order to explain why most of it can be detected in esterified form. Here we report the first experimental evidence that LCAT is capable of esterifying 24-OH Ch in cerebrospinal fluid (CSF). This result comes from experiments of competition between 24-OH Ch and Ch for LCAT and experiments of in vitro production of 24-OH Ch esters. In detail, increasing amounts of 24-OH Ch, in a standard assay of LCAT activity, decreased Ch esterification. Also, purified 24-OH Ch was incubated in a mixture containing CSF as LCAT source, ApoE, and a fluorescent phospholipid (as acyl chain donor). 24-OH Ch esters level, detected by fluorescence, was found increased in time. So, it can be excluded that 24-OH Ch esters found in CSF are produced by ACAT, and released by necrotic cells. Further experiments are required to shed light on how LCAT function on 24-OH Ch may influence brain Ch homeostasis and recovery from neuronal injury.