24S-Hydroxycholesterol (24OHC) and 27-hydroxycholesterol (27OHC) are two structurally similar oxysterols of different origins--the former almost exclusively formed in the brain and the latter formed to a lesser extent in the brain than in most other organs. HYPOTHESIS TO BE TESTED: Neuronal damage and/or demyelination causes increased flux of 24OHC from the brain into the cerebrospinal fluid (CSF), whereas a defect blood-brain barrier causes increased flux of 27OHC from the circulation into the CSF. Isotope dilution-mass spectrometry was used to assay the two oxysterols in CSF and plasma from more than 250 patients with different neurological and geriatric diseases. The CSF-levels of the two oxysterols were much more affected by the different diseases than the plasma levels. Patients with active demyelinating diseases had increased levels of 24OHC in CSF with a relatively high 24OHC/27OHC ratio. Patients with meningitis in general had high levels of both steroids with a low 24OHC/27OHC ratio. Patients with Alzheimer's disease had slightly increased levels of 24OHC in CSF with less increase in 27OHC. Patients with multiple sclerosis had a tendency to have higher levels of 24OHC during active periods with a high 24OHC/ 27OHC ratio. Measurements of the two oxysterols in CSF and plasma may add significantly to existing biochemical methods for evaluation of neurological diseases.